Hla g-modified cells and methods

ABSTRACT

Disclosed herein are methods for producing genetically modified cells expressing HLA-G (e.g., cell surface HLA-G) persistently, and nucleic acid compositions useful for generating such genetically modified cells. Also disclosed are cell therapy methods that utilize genetically modified cells that express HLA-G persistently. The HLA-G genetic modifications described herein provide the cells with characteristics of reduced immunogenicity and/or improved immunosuppression, such that these cells have the promise of being universal or improved donor cells for transplants, cellular and tissue regeneration or reconstruction, and other therapies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/608,004 filed Jan. 28, 2015, which is a continuation of InternationalPatent Application No. PCT/US2013/052767 filed Jul. 30, 2013, whichclaims the benefit of and priority to U.S. Provisional PatentApplication Ser. No. 61/677,739, filed Jul. 31, 2012, the disclosure ofeach of which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 16, 2017, isnamed 2017-06-16_126_US3_Sequence_Listing.txt and is 10,153 bytes insize.

BACKGROUND OF THE INVENTION

Regenerative medicine in the form of cell transplantation is one of themost promising therapeutic approaches for the treatment of intractablemedical conditions such as diabetes, heart disease, andneurodegenerative diseases. However, a major hurdle towards implementingcell transplantation in the clinic is immune rejection of donor cells,especially when these are derived from a foreign host. While it ispossible to address immune rejection, in part, by administeringimmunosuppressant drugs, these entail severe adverse side effects. Thus,there is an ongoing need to develop improved technologies for celltransplantation therapies.

SUMMARY OF THE INVENTION

Disclosed herein are cell-based compositions and methods for celltransplantation therapy based on the long-term forced expression of atleast an exogenous HLA-G protein in donor cells to be transplanted intoa subject in need of such donor cells. The invention provides data thatshows that cells (whether pluripotent or differentiated) modified toexpress exogenous HLA-G in the manner described herein have reducedimmunogenicity and/or increased immunosuppression. The reducedimmunogenicity and/or increased suppression abilities provided by theHLA-G genetic modification are stable and persist over long periods oftime, including through processes of differentiation. The implication isthat the HLA-G modified cells of the invention can serve as universaldonor cells or tissue (i.e., reducing or eliminating the requirement formatching the type of classical human leukocyte antigen (HLA) class I andclass II molecules between donor cells and the recipient) for numerousinjuries, diseases, or disorders.

Accordingly, in one aspect described herein is a genetically modifiedmammalian cell (an HLA-G modified cell) that has reduced immunogenicityand/or is capable of providing increased immunosuppression in anallogeneic recipient as compared to the mammalian cell without saidgenetic modification, where (i) the genetically modified mammalian cellcomprises: (a) an exogenous nucleic acid (e.g., an expression vector)comprising a nucleic acid sequence encoding an HLA-G protein having anamino acid sequence at least 85% identical to human HLA-G, andcomprising one or more amino acid mutations that reduce retention ofHLA-G in the endoplasmic reticulum-golgi recycling pathway, and/or (b) a3′ UTR (untranslated region) sequence that does not contain microRNAbinding sites such as SEQ ID NO:3 or a sequence that does not compriseSEQ ID NO:4; and (ii) the encoded HLA-G protein is expressed by thegenetically modified mammalian cell for at least seven weeks (e.g., atleast 20 weeks or at least 50 weeks).

In other embodiments, the invention provides a genetically modifiedmammalian cell that has reduced immunogenicity and/or improvedimmunosuppression as compared to the mammalian cell without said geneticmodification, wherein: (i) the genetically modified mammalian cellcomprises an exogenous nucleic acid comprising: (a) a nucleic acidsequence (such as SEQ ID NO:2) encoding an HLA-G protein having an aminoacid sequence at least 95% identical to consensus wild-type human HLA-G(such as SEQ ID NO:1), and comprising one or more amino acid mutationsthat reduce retention of HLA-G in the endoplasmic reticulum-golgirecycling pathway; and (b) a 3′ untranslated region (UTR) (such as SEQID NO:3) that is at least 85% identical to the 3′ untranslated regionsequence of the consensus wild-type human HLA-G gene and does notcomprise SEQ ID NO:4; and (ii) the encoded HLA-G protein is expressed bythe genetically modified mammalian cell for at least seven weeks.

In some embodiments, a genetically modified cell has reducedimmunogenicity and/or improved immunosuppression if it shows: (1) areduction of NK-92 cytotoxicity of the genetically modified cell ascompared to the mammalian cell without said genetic modification, (2) areduction of in vitro peripheral blood mononuclear cell proliferation ofthe genetically modified cell as compared to the mammalian cell withoutsaid genetic modification, and/or (3) an increase in the size and weightof tumor formation by the genetically modified cell as compared to themammalian cell without said genetic modification in humanized NSG mice.

In some embodiments, the genetically modified mammalian cell does nothave matches (i.e., same allele(s)) in one or more HLA antigens ascompared to the allogeneic recipient, wherein the HLA antigens areselected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-DP,HLA-DQ, and HLA-DR. In certain embodiments, the genetically modifiedmammalian cell only has 1, 2, 3, 4, or 5 matches in one or more HLAantigens as compared to the allogeneic recipient, wherein the HLAantigens are selected from the group consisting of HLA-A, HLA-B, HLA-C,HLA-DP, HLA-DQ, and HLA-DR. In one embodiment, the genetically modifiedmammalian cell has no matches in with the allogeneic recipient withrespect to HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and HLA-DR.

In some embodiments, the genetically modified cell comprises an HLA-Gtransgene without one or more amino acid mutations that reduce retentionof HLA-G in the endoplasmic reticulum-golgi recycling pathway (i.e., anHLA-G wild-type consensus sequence such as SEQ ID NO:1), but has anHLA-G transgene that comprises a 3′ UTR (untranslated region) sequencethat does not contain microRNA binding sites such as SEQ ID NO:3, or asequence that does not comprise SEQ ID NO:4.

In some embodiments, the one or more mutations that reduce retention ofHLA-G in the endoplasmic reticulum-golgi recycling pathway include adi-Lysine (KK) motif mutation. In some embodiments, the KK motifmutation incudes a K334A mutation, a K335A mutation, or both mutations.

In some embodiments, the exogenous nucleic acid to be expressed in thegenetically modified cell includes a 3′ UTR sequence that does notcontain SEQ ID NO.4. In one embodiment, where the 3′ UTR sequence of theexogenous nucleic acid does not include SEQ ID NO:4, the nucleic acidsequence contains SEQ ID NO:3.

In some embodiments, the expressed HLA-G is present on the cell surfaceof the genetically modified mammalian cell.

In some embodiments, the genetically modified mammalian cell is a humancell, a mouse cell, a rat cell, a monkey cell, or a pig cell.

In some embodiments, the genetically modified mammalian cell is a stemcell, a progenitor cell, or a cell obtained by directed differentiationof the stem cell or the progenitor cell. In some embodiments, thegenetically modified mammalian cell is a cell that was alreadydifferentiated (whether naturally or in vitro) prior to introduction ofan exogenous HLA-G transgene. In some embodiments, the geneticallymodified mammalian cell is a stem cell (e.g., a pluripotent stem cell).In some embodiments, where the genetically modified mammalian cell is astem cell, the stem cell is an embryonic stem cell, an inducedpluripotent stem cell, or a totipotent stem cell. In one embodiment, thegenetically modified mammalian cell is an embryonic stem cell. Inanother embodiment, the genetically modified mammalian cell is aninduced pluripotent stem cell. In a further embodiment, the geneticallymodified mammalian cell is not of an immune system cell type. In anotherembodiment, the genetically modified mammalian cell is a cell obtainedby in vitro differentiation of a stem cell or a progenitor cell whereinthe stem cell or progenitor cell is genetically modified and thendifferentiated in vitro.

In other embodiments, the genetically modified cell is a fullydifferentiated cell, an epidermal progenitor cell, a pancreaticprogenitor cell, a hematopoietic stem cell, a cell obtained bydifferentiation of the pluripotent stem cell, a keratinocyte, afibroblast, a mesenchymal stem cell, a cardiomyocyte, a neural stemcell, a neuron, an astrocyte, or a pancreatic β cell progenitor.

In some embodiments, where the exogenous nucleic acid in the geneticallymodified mammalian cell is an expression vector, the expression vectoris a transposon vector or a retroviral vector. In some embodiments,where the exogenous nucleic acid is an expression vector, the expressionvector is a targeting vector, and the genetically modified mammaliancell was obtained by homologous recombination of the targeting vector.In some embodiments, the expression vector may further include a nucleicacid sequence encoding a reporter protein such as green fluorescentprotein (GFP).

In some embodiments, the exogenous nucleic acid also includes a nucleicacid sequence that (i) is at least 85% identical to the 3′ untranslatedregion sequence of the human, HLA-G gene; and (ii) comprises at leastone mutation that inhibits binding of a cognate microRNA to the mutatedsite within an mRNA comprising the mutated binding site within its 3′untranslated region. In one embodiment, such a nucleic acid sequencecomprises SEQ ID NO:3.

In some embodiments an artificial tissue is provided that contains thegenetically modified cell.

In another aspect provided herein is an isolated nucleic acid thatincludes (i) a first nucleic acid sequence that encodes an amino acidsequence at least 85% identical to human, HLA-G; and (ii) a secondnucleic acid sequence that is at least 85% identical to the 3′untranslated region sequence of the human HLA-G gene and operably linkedto the first nucleic acid sequence, where the amino acid sequencecomprises a mutation that reduces retention of HLA-G in the endoplasmicreticulum-golgi recycling pathway, and the second nucleic acid sequencecomprises at least one mutation that inhibits binding of a cognatemicroRNA to an mRNA comprising the mutated binding site within its 3′untranslated region.

In some embodiments, the 3′ untranslated region sequence of the isolatednucleic acid does not comprise SEQ ID NO:4. In one embodiment, where the3′ untranslated region sequence does not comprise SEQ ID NO:4, the 3′untranslated region sequence comprises SEQ ID NO:3.

In some embodiments, a mammalian expression vector is provided thatincludes the isolated nucleic acid and a promoter operably linked to thefirst nucleic acid sequence, wherein the promoter is not silenced in astem cell. In some embodiments, the promoter contains the nucleic acidsequence of the Chinese hamster EF-1α (CHEF-1α) promoter or human EF-1αpromoter. In one embodiment, the CHEF-1α promoter comprises SEQ ID NO:6.In other embodiments, the promoter used to drive expression of an HLA-Gtransgene is a tissue or cell type-selective promoter. In someembodiments, the mammalian expression vector includes comprising a thirdnucleic acid sequence encoding a reporter protein. In some embodiments,the mammalian expression vector is a transposon vector. In someembodiments, a genetically modified mammalian cell is provided thatcontains the mammalian expression vector.

In some embodiments, an isolated nucleic acid is provided thatcomprises: (i) a first nucleic acid sequence that encodes an amino acidsequence at least 95% identical to human HLA-G, wherein the amino acidsequence comprises a mutation that reduces retention of HLA-G in theendoplasmic reticulum-golgi recycling pathway; and (ii) a second nucleicacid sequence that is at least 95% identical to the 3′ untranslatedregion sequence of the human HLA-G gene and operably linked to the firstnucleic acid sequence, wherein the second nucleic acid sequencecomprises at least one mutation that inhibits binding of a cognatemicroRNA to an mRNA comprising the mutated binding site within its 3′untranslated region. In one embodiment, the first nucleic acid sequenceencodes an amino acid sequence of SEQ ID NO:2. In another embodiment,the second nucleic acid sequence does not comprise SEQ ID NO:4. In oneembodiment, the second nucleic acid sequence comprises SEQ ID NO:3. Inanother embodiment, a mammalian expression vector is provided thatcomprises said first and second nucleic acid sequences, and furthercomprises a promoter operably linked to the first nucleic acid sequence,wherein the promoter is not silenced in a stem cell or a in a cellgenerated by differentiation of the stem cell. Such a promoter cancomprise the nucleic acid sequence of the Chinese hamster EF-1αpromoter. In another embodiment, the mammalian expression vector furthercomprises a nucleic acid sequence encoding a reporter protein. Inanother embodiment, the mammalian expression vector is a transposonvector. In another embodiment, the mammalian expression vector comprisesall of the elements shown in FIG. 1.

In one embodiment, a mammalian expression vector is provided thatcomprises: (a) a Chinese hamster EF-1 α promoter, (b) a nucleic acidsequence that is operably linked to the promoter and that encodes humanHLA-G with an amino acid sequence of SEQ ID NO:2, and (c) a 3′UTRsequence comprising SEQ ID NO:3. In some embodiments, a geneticallymodified mammalian cell is provided that comprises such an expressionvector.

In various embodiments, HLA-G modified mammalian cells (e.g., humanHLA-G modified cells) are administered to a subject suffering from anyof a number of conditions including, but not limited to cardiovasculardisease, eye disease (e.g., macular degeneration), auditory disease,(e.g., deafness), diabetes, neurodegenerative disease, Alzheimer'sDisease, Parkinson's Disease, multiple sclerosis, osteoporosis, liverdisease, kidney disease, autoimmune disease, arthritis, gum disease, adental condition, or a proliferative disorder (e.g., a cancer). In othercases, the subject is suffering from, or at high risk of suffering from,an acute health condition, e.g., stroke, spinal cord injury, burn, or awound. In other cases, the subject is suffering from loss of tissue suchas lipatrophy or aging-related losses in collagen. In other cases, thesubject suffers from a non-healing ulcer, or is need for an agent toassist in closure of defects like hypospadias and epispadias. In othercases, the subject is in need for a permanent or temporary skin graftfor wound healing or for skin substitutes.

In some embodiments, the invention provides a universal method ofcellular or tissue repair or regeneration to a subject in need thereof,the method comprising injecting or grafting to the subject a cellular ortissue composition comprising a population of enhanced HLA-G (“eHLA-G”)modified cells, wherein the subject has at least one mismatchedclassical HLA class I or HLA class II molecule as compared to thepopulation of eHLA-G modified cells, and wherein the population ofeHLA-G modified cells exhibits reduced immunogenicity and/or improvedimmunosuppression as compared to cells of the same-type without theeHLA-G modification. The reduced immunogenicity and/or improvedimmunosuppression can be determined, for example, by comparing theeHLA-G modified cell to a control cell of the same type without theeHLA-G modification in an NK-92 cytotoxicity assay, a humanized NSGtumor growth assay, and/or a PBMC proliferation assay. In oneembodiment, the population of genetically modified cells comprises apopulation of eHLA-G genetically modified human dermal fibroblasts. Inanother embodiment, the population of genetically modified cellscomprises a population of eHLA-G genetically modified human epidermalprogenitors. In another embodiment, the population of geneticallymodified cells comprises a population of eHLA-G genetically modifiedhuman mesenchymal stem cells. In another embodiment, the population ofgenetically modified cells comprises a population of eHLA-G geneticallymodified human embryonic stem (ES) cells. In another embodiment, thepopulation of genetically modified cells comprises a population of celldifferentiated in vitro from eHLA-G genetically modified human embryonicstem cells. In other embodiments, the population of genetically modifiedcells are not rejected by the subject's immune system for at least 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 24, 36, 48, or 52 weeks.

In another embodiment, the invention provides a method for regeneratingskin to a subject in need thereof, the method comprising injecting apopulation of eHLA-G modified dermal fibroblasts and/or eHLA-G modifiedembryonic epidermal progenitors to a site of skin injury on the subject,wherein the subject has at least one mismatched classical HLA class I orHLA class II molecule as compared to the population of eHLA-G modifieddermal fibroblasts and/or eHLA-G modified embryonic epidermalprogenitors.

In another embodiment, a cell therapy method is provided that comprisesadministering to a subject in need thereof a population of geneticallymodified mammalian cells comprising an exogenous human β2-microglobulin(β2m) molecule and an eHLA-G transgene of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic depiction of a non-limiting embodiment of anenhanced HLA-G (eHLA-G) transgene expression transposon vectorcontaining a selection marker (neomycin phosphotransferase) driven by aphosphoglycerate kinase (PGK) promoter; “INS” flanking insulatorelements; a Chinese Hamster EF-1α promoter driving expression of eHLA-G;a PGK promoter driving expression of EGFP; and 5′ terminal repeat (TR)transposition elements. An eHLA-G transgene contains a combination ofmutations and/or non-coding elements (such as, for example, a promoteror modified 3′ UTR) that enhance expression, particularly cell surfaceexpression, of the HLA-G protein. For the experiments described herein,the eHLA-G transgene comprised the above elements, and more specificallyincluded a human HLA-G coding sequence as listed in SEQ ID NO:2 with 1)mutations of HLA-G's ER retrieval motif (K334A/K335A); and 2) mutationof HLA-G's 3′ UTR microRNA binding sites, wherein this modified 3′ UTRhad the sequence as listed in SEQ ID NO:3. See Example 3 for furtherdescription.

FIG. 2 shows a schematic depiction of a non-limiting embodiment of atransposase expression helper vector used to drive genomic integrationof a co-transfected transposon expression vector.

FIG. 3 shows fluorescence micrographs of human ES cell colonies atvarious times after stable transfection with a transposon expressionvector driving expression of eHLA-G and EGFP.

FIG. 4 (Top Panel) shows a bar graph depicting the relationship betweenthe starting number of hES cells transfected with an EGFP-containingexpression vector and the fraction of EGFP⁺ cells detected in the cellpopulation at 10 days post-transfection. The numbers on the x-axis arethe number of GFP⁺ colonies per 5×10⁵ cells. The numbers on the y-axisare the cell number. (Bottom Panel) shows a bar graph depicting therelationship between various transposon vectors (using differentpromoters) and the fraction of EGFP⁺ cells detected in the cellpopulation. The numbers on the x-axis are the number of GFP⁺ coloniesper 5×10⁵ cells. For the bottom panel, from left to right: control(con), GFP-puro (size of transfected vector is 7.3 kb); eHLA-G(MSCV)-GFP-puro (size of transfected vector is 8.6 kb); and eHLA-G(EF-1α)-GFP-puro (size of transfected vector is 9.2 kb). AdditionaleHLA-G (EF-1α)-GFP-puro transfection efficiency experiments (not shown)were conducted, which indicated that at 10 days post-transfection, thehighest efficiencies (˜500 GFP⁺ colonies per 5×10⁵ cells) were obtainedin solution V and program B16 after selection with puromycin for 10days.

FIG. 5 shows a series of immunofluorescence micrographs showingexpression in HLA-G modified human ES cells of HLA-G expression and DAPIstaining(top images); Oct 3/4 and DAPI (middle images); and SSEA-4 andDAPI (bottom images).

FIG. 6 (Top Panel) shows a flow cytometry scattergram showing thedistribution of SSEA-4⁺/GFP⁺ double-positive cells in a population ofeHLA-G-modified hES cells. (Lower Panel) shows the distribution of Oct3/4⁺ cells in the population of eHLA-G-modified hES cells. SSEA-4 andOct 3/4 are pluripotency markers. This data, along with FIG. 5, indicatethat eHLA-G-modified hES cells maintained their characteristicself-renewal pluripotency markers. Additionally, eHLA-G(EF-1α)-GFP-hESCsmaintained their pluripotency and normal karyotype in vivo. HumanizedNSG mice were injected subcutaneously with eHLA-G(EF-1α)-GFP-hESCs, andteratoma formation was observed, indicating that theinjected/transplanted cells were not rejected as hESCs exhibited reducedimmunogenicity and/or increased immunosuppression. The karyotype of theteratoma cells were normal.

FIG. 7 (Top Panel) shows phase contrast photomicrographs of Wildtype(left picture) and eHLA-G modified hESC-generated (right picture)embryoid bodies (EBs) at day 15. (Bottom Panel) shows a fluorescencemicrograph of the EBs shown in the Top Panel. No GFP signal is detectedin the wildtype hESC EB, whereas strong GFP expression is detected inboth of the eHLA-G modified hESC EBs. This data indicates that eHLA-G⁺hESCs maintain EB formation.

FIG. 8 shows that eHLA-G⁺ hESCs are silencing resistant. (Top Panel)shows a flow cytometry distribution histogram for the expression of GFPin a eHLA-G modified hESC-line, which demonstrated similar strongexpression of GFP after 6 and 16 passages. (Bottom Panel) shows a flowcytometry distribution histogram for the expression of HLA-G in the samehESC line, again showing persistent expression of the eHLA-G transgeneat both passage 6 and 16.

FIG. 9A (Top Panel) shows flow cytometry distribution histograms fortotal (intracellular) expression HLA-G in wildtype (left histogram),GFP-modified (middle histogram), and eHLA-G(EF-1α)-GFP modified hESCs(right histogram). (Bottom Panel) shows flow cytometry distributionhistograms for surface expression HLA-G in wildtype (left histogram),GFP-modified (middle histogram), and eHLA-G (EF-1α)-GFP modified hESCs(right histogram). FIG. 9B (Top Panel) shows a flow cytometrydistribution histogram for total expression HLA-G in eHLA-G(MSCV)-GFPmodified hESCs. FIG. 9B (Bottom Panel) shows surface HLA-G expressioneHLA-G(MSCV)-GFP modified hESCs. These data indicate HLA-G is highlyexpressed when the transgene is operably linked to the EF-1α promoter,in contrast the minimal expression when the transgene is under controlof the MSCV promoter. See also FIG. 15 for additional data showing thatHLA-G transgene expression is significantly influenced by promoteractivity.

FIG. 10 shows that expression of HLA class I and class II is similar onwildtype and eHLA-G+ hESCs. The table compares expression levels ofvarious HLA variants and β2 microglobulin in wildtype versus HLA-Gmodified hESCs.

FIG. 11 shows that NK92 cytotoxicity effect is greatly suppressed byeHLA-G⁺ hESCs. (See Example 6 for further description.) The figure showsa bar graph illustrating the results of an NK92 cell cytotoxicity assay.The 1:10 and 1:30 values indicate the ratio of effector (NK92) to targetcells (GFP transgene alone control wild-type cells or eHLA-G-GFPtransgene modified cells). This is an in vitro assay to determine theimmunogenicity of eHLA-G modified hESCs (black bars; eHLA-G-GFP)compared to wildtype hESCs (grey bars; GFP). eHLA-G modified hESCsexhibit substantially reduced cytotoxicity in the presence of NK92 cellsas compared to the case for wildtype hESCs. This data indicates thatexogenous HLA-G expression can provide improved donor capabilities forsuch genetically modified cells, as reduced cytotoxicity in the presenceof NK92 cells shows that such genetically modified cells have reducedimmunogenicity and/or improved immunosuppression. Results are theaverage of four experiments. See Example 6 for further description.

FIG. 12 shows a series of bar graphs depicting the time course of geneexpression levels of several pluripotency markers and epidermalprogenitor markers during directed differentiation of hESCs in vitrointo embryonic epidermal progenitors (EEPs). The y-axis values arerelative mRNA expression levels as determined by semi-quantitativeRT-PCR. The x-axis values are the days at which expression was assessed.See Example 2 for further description. The FIG. 12 data indicates thatthe epidermal differentiation markers K14, Tap63, and ΔNp63 weregradually enhanced during differentiation. In data not shown here,immunofluorescence studies of K14 and additional epidermal markers p63,CD29, and CD49f were conducted. Differentiated eHLA-G(EF-1a)-GFP hEEPswere positive for K14, p63, CD29, and CD49f protein expression asindicated by immunofluorescence.

FIG. 13 (Top Panel) shows time course of eHLA-G transgene expression inan hESC line stably transfected with EF-1α promoter-driven expression ofeHLA-G. (Bottom Panel) shows a comparison of the time course (black barsindicate at 0 days; horizontal striped bars indicate at 7 days; and greybars indicate at 14 days) of HLA-G (mRNA) expression in a GFP-modifiedhESC line (negative control), an MSCV-promoter driven eHLA-G hESC line,and in an EF-1α driven eHLA-G hESC line. Note the higher and morepersistent level of eHLA-G expression driven by the EF-1α promoter.

FIG. 14 (Effect of K562-HLA-G1 14 base pair (bp) insertion/deletionpolymorphism on cytotoxic activity of NK cells.) The figure shows a bargraph comparing NK cell-mediated cytotoxicity (% specific lysis) onwildtype K562 cells (grey bars), K562 cells expressing an HLA-G variantwith a 14 bp insertion in the 3′ UTR (Ins14bp) (black bars), and K562cells expressing an HLA-G variant with a 14 bp deletion in the 3′ UTR(Del 14bp) (grey bars). There are four sets of data with differenteffector (NK cell) to target cell (K562 cells) ratios.

FIG. 15 provides data that indicates that HLA-G transgene expression issignificantly influenced by promoter activity. RT-PCR shows that bothGFP and HLA-G transcripts were highly expressed in eHLA-G(EF-1α)-GFP-hESC cell lines. Immunofluorescence also shows that both GFPand HLA-G proteins were highly expressed in HLA-G (EF-1α)-GFP-hESC celllines (not shown here). However, HLA-G transcripts or proteins wererarely detected in HLA-G (pMSCV)-GFP-hESC lines, whether by RT-PCR orimmunofluorescence, even though GFP expression was high in these cells.(Data not shown here.) Thus, EF-1α promoters are preferred in certainembodiments of HLA-G transgene expression.

FIG. 16. Purified hEEPs exhibited homologous keratinocyte morphology asshown by phase contrast microscopy. eHLA-G-GFP-hEEP clones 18 and 21were differentiated from modified eHLA-G(EF-1α)-GFP-hESCs as describedin Example 2.

FIG. 17. The stability of the HLA-G transgene in differentiated hEEPswas confirmed by flow cytometry. Both HLA-G total expression (toppanels) and surface expression (bottom panels) was robust fordifferentiated eHLA-G(EF-1a)-GFP-hEEPs (greater than 90% of cells) ascompared to control cells with no exogenous HLA-G (GFP only hEEPs) andwild-type hEEPs.

FIG. 18. The results of FIG. 11 were repeated and confirmed inadditional NK cytotoxicity experiments. As shown, killing ofeHLA-G(EF-1α)-GFP-hESCs was reduced more than 100% as compared tocontrols hESCs that contained only a GFP transgene (no HLA-G transgene).(Note: as used herein, an “mHLA-G(EF-1α)-GFP” transgene is synonymouswith “eHLA-G(EF-1α)-GFP”.) This data shows that HLA-G transgeneexpression imparts immunosuppressive and/or reduced immunogenicitycharacteristics in hESCs. See Example 6 for further description.

FIG. 19. NK cytotoxicity experiments were conducted on hEEPsdifferentiated from hESCs. As shown, killing of eHLA(EF-1α)-GFP-hEEPswas reduced well more than 100% (about 3-fold) as compared to controlhEEPs. This data shows that HLA-G transgene expression impartsimmunosuppressive and/or reduced immunogenicity characteristics indifferentiated cells, which also shows that these enhanced functionalimmune evasion characteristics of HLA-G expression via the eHLA-Gtransgene survived the directed differentiation process. See Example 6for further description.

FIG. 20 shows the results of hESC allografts in humanized mice. The “GO”hESCs were the control wild-type hESCs that do not contain an eHLA-Gtransgene, but rather only GFP. “mG1(#1)” and “mG1(#2)” refer to twodifferent eHLA-G(EF-1α)-GFP nucleofected hESC clones. The G0, mG1(#1),and mG1(#2) tumors as shown were measured and weighed. The G0 hESCsformed a tumor with a volume of 126.9 cubic millimeters and a weight of32 milligrams. The mG1(#1) hESCs formed a tumor with a volume of 748.4cubic millimeters and a weight of 318 milligrams. The mG1(#2) hESCsformed a tumor with a volume of 1116.7 cubic millimeters and a weight of675 milligrams. See Example 7 for further description.

FIG. 21 shows the averaged results of tumors from hESC allografts ontofive humanized NSG mice. Top panel shows tumor weight (mg) results. Thebottom panel shows tumor volume (cubic millimeters) results. The datashows that HLA-G nucleofected hESCs (“mG1”) formed much larger (morethan 3-fold by volume) and heavier (more than 2-fold by weight) tumorsthan wild-type hESCs (“G0”) transplanted into humanized NSG mice. Thisindicates that HLA-G transgene expression can provide reducedimmunogenicity and/or increased immunosuppression in an allograft humanenvironment (i.e., NSG humanized mice). This data, along with the NK92cytotoxicity studies, supports the general application of the eHLA-Gtransgene constructs described herein for modifying any desiredcell-type into a universal or superior allogeneic donor for therapy,transplants, tissue repair, cell and tissue substitutes, and the like.See Example 7 for further description.

FIG. 22. Human dermal fibroblasts stably transfected with theeHLA-G(EF-1α)-GFP transgene (“HFD-m1-GFP” cells) or GFP-alone controlconstruct (“HFD-G0-GFP” cells) were assessed for their ability toinhibit PBMC proliferation. As shown, the HFD-mG1-GFP clone “mG1-R1”suppressed PBMC proliferation greater than controls and other clones,indicating that exogenous HLA-G expression can provide immunosuppressionfor differentiated cells, such as fibroblasts. See Example 9 for furtherdescription, including a summary of NK-92 cytotoxicity studies withHFD-m1-GFP and controls, which shows that the eHLA-G modification tohuman dermal fibroblasts reduced their immunogenicity. Thus, these datafurther support the use of the eHLA-G transgene constructs describedherein for modifying any desired cell-type, whether pluripotent,multipotent, or fully differentiated, into a universal or superiorallogeneic donor for therapy, transplants, tissue repair, cell andtissue substitutes, and the like.

DETAILED DESCRIPTION

The present disclosure features genetically modified mammalian cellsthat express exogenous HLA-G persistently (HLA-G modified cells), aswell as nucleic acid compositions to generate such modified mammaliancells. The eHLA-G genetic modifications described herein provide thecells with characteristics of reduced immunogenicity and/or improvedimmunosuppression, such that these cells have the promise of beinguniversal or improved donor cells for transplants, cellular and tissueregeneration or reconstruction, and other therapies.

I. Compositions:

A. Genetically Modified Mammalian Cells that Express Exogenous HLA-G

As described herein, a wide range of mammalian cell types that expressexogenous HLA-G (HLA-G modified cells) can be generated. Such cell typesinclude, but are not limited to, totipotent cells, embryonic stem cells(e.g., human embryonic stem cells), induced pluripotent stem cells(e.g., human induced pluripotent stem cells), multipotent stem cells,epidermal progenitor cells, mesenchymal stem cells, pancreatic β cellprogenitors, pancreatic β cells, cardiac progenitors, cardiomyocytes,hepatic progenitors, hepatocytes, muscle cell progenitors, muscle cells,kidney cells, osteoblasts, hematopoietic progenitors, dental folliclecells, hair follicle cells, retinal pigment epithelial cells, neuralstem cells, neurons, astrocytes, oligodendrocytes, inner ear cells, andfibroblasts (including human dermal fibroblast (HFD)). In someembodiments, the HLA-G modified cells are not cells having an immunesystem cell type. Such mammalian cells can be derived from one ofseveral species including, e.g., human, mouse, rat, monkey, or pig. Inessence, any cell-type can be transfected with the constructs describedherein and then tested for HLA-G expression and how such expression canimpart reduced immunogenicity and/or improved immunosuppression to themodified cell.

In some embodiments, to obtain a substantially enriched population ofHLA-G modified cells of a desired cell type, a genetically modifiedpluripotent stem cell line such as a human embryonic stem cell line, ora human induced pluripotent stem cell line, or any cell line that hasmultipotent traits including mesenchymal stem cells and immune systemprogenitor cells, that expresses HLA-G is generated and then subjectedto directed differentiation to obtain a cell population that expressesHLA-G and that is substantially enriched for a desired cell type. Insome embodiments, the substantially enriched cell population includes atleast about 2% to about 100% of the desired cell type, e.g., at leastabout 3%, 4%, 5%, 7%, 8%, 10%, 20%, 22%, 25%, 35%, 40%, 45%, 50%, 60%,70%, 75%, 80%, 85%, or another percentage of the desired cell type fromat least about 2% to about 100%. Methods for enriching cells of adesired cell type are known in the art. See, e.g., U.S. patentapplication Ser. No. 12/532,512.

Methods for obtaining human embryonic stem cells or induced pluripotentstem cells are known in the art, as described in, e.g., U.S. Pat. Nos.6,200,806 and 7,217,569 (for human embryonic stem cell derivation) andU.S. Pat. Nos. 8,048,999, 8,058,065, and 8,048,675 (for generation ofhuman induced pluripotent stem cells).

Genetically modified mammalian pluripotent or multipotent stem celllines, e.g., human embryonic stem cells or human induced pluripotentstem cells, etc., and also fully differentiated genetically modifiedmammalian, that stably express an HLA-G protein encoded by one of thenucleic acids described herein are generated by any of a number ofmethods known in the art. In some embodiments, the cell line isgenetically modified by stable transfection with one or more nucleicacid expression vectors (e.g., a plasmid vector or a minicircle vector)that include an expression cassette for expression of an HLA-G proteinas described herein, a selection marker, and optionally a reporterprotein. Examples of suitable selection markers encoded by such vectorsinclude proteins that confer resistance to a selection agent. Suchproteins and their corresponding selection agents include, withoutlimitation, puromycin N-acetyltransferase (puromycin), hygromycinphosphotransferase (hygromycin), blasticidin-S-deaminase (blasticidin),and neomycin phosphotransferase (neomycin). Selection with theappropriate selection agent may last for at least about 3 to about 14days, e.g., about 4, 5, 6, 7, 8, 9, 10, 12, 13, or another period fromabout 3 to about 14 days until resistant colonies are apparent.

Examples of suitable fluorescent reporter proteins include, but are notlimited to, EGFP and its variants such as YFP, Cyan, and dEGFPs; DS-Red,monomeric Orange, the far-red fluorescent protein “Katushka” (Shcherboet al (2007), Nat Methods, 4:741-746), or variants of any of theforegoing. In other embodiments, the reporter is an enzyme that convertsa substrate that, in the process, yields a detectable signal, e.g., afluorescent or luminescent signal in the presence of a fluorogenic orluminogenic substrate, respectively. For example, in some embodiments,the selection marker enzyme comprises the amino acid sequence of aluciferase, e.g., a firefly luciferase, click beetle luciferase, orRenilla luciferase. Luciferase activity can be detected by providing anappropriate luminogenic substrate, e.g., firefly luciferin for fireflyluciferase or coelenterazine for Renilla luciferase. Luciferase activityin the presence of an appropriate substrate can be quantified byluminometry to assay total luciferase activity of whole cell populationsin culture dish wells, or, alternatively, luciferase activity ofindividual cells or colonies can be detected by use of a microscope incombination with a photon counting camera. Details of luciferase assays,including high-throughput methods, are disclosed in, e.g., U.S. Pat.Nos. 5,650,135, 5,744,320, and 6,982,431. In other embodiments, thereporter enzyme comprises the amino acid sequence of a modifiedbeta-lactamase, the expression of which can be detected and quantifiedin living cells by a ratiometric fluorescence assay for breakdown offluorogenic beta-lactam substrates as described in, e.g., U.S. Pat. Nos.5,741,657, 6,031,094, and U.S. Patent Publication No. 20070184513. Seealso Qureshi (2007), Biotechniques, 42(1):91-96 for a review. Othersuitable reporter enzymes include, but are not limited to, the Halo-Tag®hydrolase (Promega, Madison, Wis., as described in, e.g., U.S. Pat. No.7,238,842 and Patent Publication Nos. 20080026407 and 20080145882) andbeta.-galactosidase.

In some embodiments, the genetically modified mammalian cells are alsogenetically modified to express a human β2 microglobulin (GenBankAccession No. AY187687.1). Without wishing to be bound by theory, it isbelieved that expression of human β2 microglobulin will enhance surfaceexpression of transgenic HLA-G in the genetically modified mammaliancells.

In some embodiments, the nucleic acid expression vector is a transposonvector that includes transposition elements that facilitate integrationof the transposition of the transposon vector into a host genome whenintroduced into a host cell in the presence of a cognate transposase(e.g., the piggyBAC transposase), as described in, e.g., U.S. patentapplication Ser. No. 12/728,943. Transposon expression vectors (e.g.,PiggyBac vectors) as well as transposase expression vectors arecommercially available from, e.g., System Biosciences (Mountain View,Calif.). In some embodiments, where a transposon vector is used, noselection marker or reporter protein expression cassette are necessaryto generate a stably transfected, HLA-G modified cell line, as theefficiency of transfection by such vectors is sufficiently high toobviate the need for a selection marker. Alternatively, the expressionvector may be a targeting vector, which allows site-specific integrationof the eHLA-G transgene in the host cell genome. The design and use oftargeting vectors is routine in the art as exemplified by U.S. Pat. No.5,464,764.

Methods for preparation of transfection-grade nucleic acid expressionvectors and transfection methods are well established. See, e.g.,Sambrook and Russell (2001), “Molecular Cloning: A Laboratory Manual,”3rd ed., (CSHL Press); and Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (2005), 9.1-9.14. Examples of high efficiencytransfection efficiency methods include “nucleofection,” as describedin, e.g., Trompeter (2003), J Immunol. Methods, 274(1-2):245-256, and inU.S. Pat. Nos. 7,332,332, 8,003,389; 8,039,259, and 8,192,990 9,transfection with lipid-based transfection reagents such as Fugene®(Roche), DOTAP, and Lipofectamine™ (Invitrogen).

In other embodiments, genetically modified cells, e.g., differentiated,multipotent, pluripotent, or totipotent stem cell lines, are generatedby transduction with a recombinant virus. Examples of suitablerecombinant viruses include, but are not limited to, retroviruses(including lentiviruses); adenoviruses; and adeno-associated viruses.Often, the recombinant retrovirus is murine moloney leukemia virus(MMLV), but other recombinant retroviruses may also be used, e.g., AvianLeukosis Virus, Bovine Leukemia Virus, Murine Leukemia Virus (MLV),Mink-Cell focus-Inducing Virus, Murine Sarcoma Virus,Reticuloendotheliosis virus, Gibbon Ape Leukemia Virus, Mason PfizerMonkey Virus, or Rous Sarcoma Virus, see, e.g., U.S. Pat. No. 6,333,195.

In other cases, the recombinant retrovirus is a lentivirus (e.g., HumanImmunodeficiency Virus-1 (HIV-1); Simian Immunodeficiency Virus (SIV);or Feline Immunodeficiency Virus (FIV)), See, e.g., Johnston et al.,(1999), Journal of Virology, 73(6):4991-5000 (FIV); Negre et al.,(2002), Current Topics in Microbiology and Immunology, 261:53-74 (SIV);Naldini et al., (1996), Science, 272:263-267 (HIV).

The recombinant retrovirus may comprise a viral polypeptide (e.g.,retroviral env) to aid entry into the target cell. Such viralpolypeptides are well-established in the art, see, e.g., U.S. Pat. No.5,449,614. The viral polypeptide may be an amphotropic viralpolypeptide, e.g., amphotropic env, that aids entry into cells derivedfrom multiple species, including cells outside of the original hostspecies. See, e.g., id. The viral polypeptide may be a xenotropic viralpolypeptide that aids entry into cells outside of the original hostspecies. See, e.g., id. In some embodiments, the viral polypeptide is anecotropic viral polypeptide, e.g., ecotropic env, that aids entry intocells of the original host species. See, e.g., id.

Viral transduction of cells may be accomplished by any method known inthe art. e.g., Palsson, B., et al., (1995), WO95/10619; Morling, F. J.et al., (1995), Gene Therapy, 2:504-508; Gopp et al., (2006), MethodsEnzymol, 420:64-81. For example, the infection may be accomplished byspin-infection or “spinoculation” methods that involve subjecting thecells to centrifugation during the period closely following the additionof virus to the cells. In some cases, virus may be concentrated prior tothe infection, e.g., by ultracentrifugation.

The multiplicity of infection (m.o.i.) used to transduce the cells to begenetically modified can range from about 1 m.o.i. to about 50 m.o.i.,e.g., about 1 m.o.i., about 5 m.o.i., about 7.5; m.o.i., about 10m.o.i., about 15 m.o.i., about 20 m.o.i., about 30 m.o.i., about 40m.o.i., or about 50 m.o.i.

Methods for generating various cell types from pluripotent stem cells(e.g., human embryonic stem cells or human induced pluripotent stemcells) by directed differentiation are known in the art, as describedin, e.g., U.S. Pat. Nos. 7,955,849, 7,763,466, 7,264,968; and U.S.patent application Ser. Nos. 12/179,462, and 12/187,543.

In one exemplary embodiment, human embryonic epidermal progenitors(hEEPs) are derived from a human pluripotent stem cell line, e.g., humanembryonic stem cells or human induced pluripotent stem cells as follows.

Pluripotent stem cells are maintained in Embryonic Stem Cell (ESC)growth medium containing DMEM/F12 (1:1) supplemented with 20% knockoutserum replacement, 0.1 mM MEM non-essential amino acids, 1 mM GlutaMax,0.1 mM β-mercaptoethanol (Sigma). ESC growth medium is conditioned byplating mitotically inactivated mouse embryonic fibroblasts (MEFs)(CF-1, ATCC) at a density of 5×10⁴ cells/cm² and incubating for 18-24hours. After conditioning, 4 ng/ml bFGF is added and the completelyconditioned medium is sterile filtered. hESCs are subcultured every 5-6days (at a 1:3 or 1:4 split) on Matrigel® (BD Biosciences)-coated platesusing 1 mg/ml Dispase to remove cell colonies. hEEPs Differentiation ofpluripotent stem cells into K14⁺/p63⁺ hEEPs, by first culturing thepluripotent stem cells in 6-well plates for 4 days in ESC growth mediumand then changing over to 2 ml/well of differentiation medium, comprisedof unconditioned hESC growth medium containing 1 μM all-trans retinoicacid (Sigma) and 25 ng/ml BMP4. After daily medium changes for 7 days,cells are treated with dispase, centrifuged, and resuspended in definedkeratinocyte serum-free medium (DSFM) and seeded on gelatin-coatedplates at a split ratio of 1:3. DSFM will be changed every other day for3-4 weeks. Cells are then subcultured using trypsinization, centrifuged,washed, and plated at 10,000 cells per cm² on gelatin-coated tissueculture plates in DSFM. To verify that epithelial monolayers are of ≧90%purity and express K14, cells are subjected to flow cytometry accordingto the method of Metallo et al (2010), Methods Mol Biol 585:83-92. Toenhance the purity of isolated hEEPs, cells were sorted using magneticactivated cell sorting (MACS) with CD29 antibodies. About 92 percent ofCD29 MACS sorted hEEP cell culture cells differentiated fromeHLA-G(EF-1α)-GFP modified hESCs were positive for K14, a specifickeratinocyte marker.

Genetically modified mammalian cells expressing transgenic eHLA-G, asdescribed herein, have reduced immunogenicity relative to correspondingmammalian cells that do not express HLA-G. For example, immunogenicitymay be reduced by at least about 5% to about 95%, relative to acorresponding cell type that does not express exogenous HLA-G, e.g.,about 6%, 7%, 10%, 12%, 15%, 20%, 30%, 40%, 50%, 65%, 70%, 80%, 85%,90%, or another percent reduced immunogenicity relative to cells of thesame cell type that do not express exogenous HLA-G.

Methods for determining immunogenicity of cells are known in the art.For example, in some embodiments, HLA-G modified mammalian cells (e.g.,human embryonic stem cells, or differentiated cells from HLA-G modifiedembryonic stem cells, or cells that are already fully differentiatedprior to modification, etc.) or unmodified mammalian cells are culturedin the presence of an allogeneic natural killer cell line (e.g., NK-92)and then cytotoxicity to the HLA-G modified versus unmodified cells byinduced by the NK-92 cells is determined by any of a number of standardcell viability assays.

Nucleic Acids Containing Enhanced HLA-G (eHLA-G) Transgene

The isolated nucleic acids (e.g., mammalian plasmid expression vectors)used to generate HLA-modified mammalian cells, as described herein,contain an enhanced HLA-G “eHLA-G” transgene that drives increased cellsurface expression and/or secretion of HLA-G relative to cell surfaceexpression driven by a wildtype HLA-G transgene. Such a transgenetypically includes at least three distinct components: a promoter and 5′untranslated region (5′ UTR) sequence; a coding sequence; and a 3′untranslated region (3′ UTR) sequence.

In some embodiments, the promoter to be used to drive eHLA-G transgeneexpression is one capable of driving expression of the efILA-G transgenein a cell type of interest for a period of at least about seven weeks toabout 50 weeks, e.g., 8 weeks, 9 weeks, 10 weeks, 12 weeks, 15 weeks, 20weeks, 25 weeks, 30 weeks, 35 weeks, 40 weeks, 42 weeks, 45 weeks, 47weeks, 48 weeks, or another period from at least about seven weeks toabout 50 weeks. Promoters capable of driving expression for suchextended periods of time are effective in evading silencing that occursin a number of cell types including, e.g., stem cells such as embryonicstem cells, induced pluripotent stem cells, or mesenchymal stem cells.

Suitable, silencing-resistant promoters include, but are not limited to,the Chinese hamster elongation factor-1 alpha (CHEF-1α) promoter (seeRunning Deer et al (2004), Biotechnol. Prog., 20:880-889; and GenBankAccession No. AY188393.1), the M-U3/R-variant promoter of the MurineStem Cell Virus (MSCV) promoter (as described in Swindle et al (2004), JBiol Chem, 279:34-41), the phosphoglycerate kinase (PGK) promoter, thehuman β-actin promoter, and the ubiquitin C promoter.

In some embodiments, the promoter used to drive expression of an eHLA-Gtransgene is expressed in one or more desired cell types at a level thatis higher than in other cell types. One of ordinary skill in the artwill appreciate that, for example, where an HLA-G modified stem is to bedifferentiated into a particular cell type, it may be advantageous toselect a promoter that is active within or even selective for thatparticular cell type. For example, for a given tissue or celltype-selective promoter the expression level may be about two fold to100 fold higher in the desired cell type compared to another cell type,e.g., about 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 25 fold, 30 fold,40 fold, 50 fold, 70 fold, 80 fold, 90 fold, or another fold higherlevel of expression in the desired cell type compared to another celltype. Examples of tissue and/or cell type-selective promoters include,but are not limited to, the promoters for: Neuron-Specific Enolase(neuronal), Synapsin (neuronal), CamKII (forebrain neurons), HB9 (motorneurons), and Dopamine Transporter (dopaminergic neurons); GlialFibrillary Acidic Protein (astrocytes); Albumin (liver); α-Myosin HeavyChain (αMHC-cardiomyocytes); Neurogenin 3 and Pancreas-Duodenum Homeobox1 (pancreas); Keratin 14 (skin); and Bestrophin1 (retinal pigmentepithelium);

Typically the eHLA-G transgene sequence encodes an HLA-G protein thatcontains at least one to about ten point mutations relative to the human(GenBank No. NP_002118.1), or chimpanzee consensus sequence, e.g., 2, 3,4, 5, 6, 7, 8, 9, or 10 point mutations relative to the above-mentionedHLA-G protein consensus sequences.

The human consensus wildtype sequence (SEQ ID NO:1) is shown below:

MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEHLTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVA AVLWRKKSSD

In some embodiments, the at least one to about ten point mutationsincrease the level of expression of the HLA-G protein on the cellsurface of the expressing host cell by reducing retention of HLA-G inthe endoplasmic reticulum during processing and maturation of theprotein. Such mutations include, for example, an HLA-G “KK” motifmutation. See, e.g., Park et al (2001), Immunity, 15:213-224. In somecases the KK motif mutation includes a K334A mutation, a K335A mutation,or both substitution mutations. In other cases, the substitution may bemade with a different aliphatic amino (e.g., leucine) acid or anothertype of amino acid that is non-basic.

In one example, the encoded HLA-G protein has the amino acid sequence of(SEQ ID NO:2), in which K334A and K335A substitutions, relative to thewildtype sequence, have been introduced (underlined):

MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKVVAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLW RAASSD

In some embodiments, an eHLA-G transgene encodes an HLA-G protein, theamino acid sequence of which is at least 75% to 100% identical to thatof SEQ ID NO:2, e.g., 77%, 80%, 82%, 85%, 87%, 88%, 90%, 92%, 95%, 96%,97%, 98%, 99%, or another percent identical to the amino acid sequenceof SEQ ID NO:2.

The eHLA-G transgene disclosed herein also includes a 3′ untranslated(3′ UTR) region containing a number of regulatory elements affecting theexpression/translational efficiency of HLA-G transcripts. In someembodiments the eHLA-G transgene 3′ UTR sequence is a nucleic acidsequence that includes a nucleic acid sequence that is at least 75%identical to the sequence of (SEQ ID NO:3), e.g., at least 77%, 80%,82%, 85%, 87%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, or another percentidentical to the sequence of HLA-G 3′ UTR (SEQ ID NO:3):

TGTGAAACAGCTGCCCTGTGTGGGACTGAGTGGCAAGTCCCTTTGTGACTTCAAGAACCCTGACTTCTCTTTGTGCAGAGACCAGCCCAACCCTGTGCCCACCATGACCCTCTTCCTCATGCTGAACTGCATTCCTTCCCCAATCACCTTTCCTGTTCCAGAAAAGGGGCTGGGATGTCTCCGTCTCTGTCTCAAATTTGTGGTCCACTGAGCTATAACTTACTTCTGTATTAAAAT TAGAATCTGAGTG

Such sequences contain mutations (underlined) that decrease binding oftwo microRNA binding sites that result in increased expression of thetrangene-encoded HLA-G. These sequences include a deletion of a 14 basepair sequence present in exon 8 of some HLA-G alleles. This 14 base pairsequence is shown below as SEQ ID NO NO:4:

14-bp insertion sequence in HLA 3′ UTR SEQ ID NO: 4 ATTTGTTCATGCCT

SEQ ID NO:5, shown below, corresponds to SEQ ID NO:3 with the insertionof this 14 base pair sequence (lower case):

(+14 BP HLA-G 3′ UTR variant) SEQ ID NO: 5 TGTGAAACAGCTGCCCTGTGTGGGACTGAGTGGCAAGatttgttcatgcctTCCCTTTGTGACTTCAAGAACCCTGACTTCTCTTTGTGCAGAGACCAGCCCAACCCTGTGCCCACCATGACCCTCTTCCTCATGCTGAACTGCATTCCTTCCCCAATCACCTTTCCTGTTCCAGAAAAGGGGCTGGGATGTCTCCGTCTCTGTCTCAAATTTGTGGTCCACTGAGCTATAACTTACTTCTGTATTA AAATTAGAATCTGAGTG

It has been reported that HLA-G alleles having a 3′ UTR includes theabove-mentioned 14 base insertion (see SEQ ID NO:4), yield a more stableHLA-G mRNA transcript. See Rouseau et al (2003), Human Immunology,64:1005-1010. Surprisingly, as one basis for some embodiments of theinstant disclosure, it was unexpectedly found that expression of theallele with a 14 base pair deletion (shown in SEQ ID NO:3) appeared tobe more immunoprotective than the allele that includes the 14 pairinsertion. See FIG. 13. While not wishing to be bound by theory, it isbelieved that the effect of the 14 bp deletion on expression levels ofHLA-G may be cell type-specific, e.g., the 14 bp deletion apparentlyenhances expression of eHLA-G in human cells, including at least hESCs,hEEPs, and human dermal fibroblast cells.

In some embodiments, the nucleic acid sequence containing the eHLA-Gtransgene also includes insulator sequences that flank the eHLA-Gexpression cassette. Insulator sequences mitigate genomic positioneffects that could spuriously affect expression of an integratedexogenous expression cassette. In some embodiments, the insulatorsequences to be used contain the chicken β-globin HS4 core insulatorsequence.

In some embodiments, the nucleic acid containing the eHLA-G transgenewill also include a selection marker as described herein. Optionally,the isolated nucleic acid containing the eHLA-G transgene may furthercontain a reporter protein as described herein.

In some embodiments, an eHLA-G-modified cell line is generated by theuse of a transposon vector that comprises an eHLA-G expression cassette,but does not contain expression cassettes for a selection marker or areporter protein. The vector is introduced to the cells to be modifiedalong with a transposase expression vector, followed by limitingdilution cloning. As the population of cells transfected is veryefficiently modified, the need to use selection markers or reporterproteins can be avoided. This is an important consideration, especiallyfor cells to be used in the context of cell therapy in human patients.The percentage of cells that is successfully modified bytransposon-based stable transfection methods can range from about 0.5%to about 50%, e.g., 1%, 2%, 3%, 5%, 7%, 8%, 15%, 20%, 22%, 30%, 40%, oranother percentage from about 0.5% to about 50% of the cellstransfected.

In some embodiments, expression of proteins encoded by a nucleic acidencoding two or more proteins is driven by separate promoters. In otherembodiments, a polycistronic expression cassette may incorporate one ormore internal ribosomal entry site (IRES) sequences between open readingframes incorporated into the polycistronic expression cassette. IRESsequences and their use are known in the art as exemplified in, e.g.,Martinez-Salas (1999), Curr Opin Biotechnol, 10(5):458-464.Alternatively, multiple open reading frames may be linked to each otherby an intervening 2A peptide sequence of the foot-and-mouth diseasevirus (F2A) or 2A-like sequences from other viruses. See, e.g., Hasegawaet al (2007), Stem Cells, 25:1707-1712 and Symczak et al (2004), NatBiotechnol, 589-594. Inclusion of the 2A peptide sequence allowspost-translational cleavage of a contiguous polypeptide containingeHLA-G and other sequences (e.g., a reporter protein sequence or aselection marker protein sequence) into separate proteins.

While identity between relatively short amino acid or nucleic acidsequences can be easily determined by visual inspection, analysis withan appropriate algorithm, typically facilitated through computersoftware, commonly is used to determine identity between longersequences. When using a sequence comparison algorithm, test andreference sequences typically are input into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters. A number of mathematical algorithms for rapidly obtainingthe optimal alignment and calculating identity between two or moresequences are known and incorporated into a number of available softwareprograms. Examples of such programs include the MATCH-BOX, MULTAIN, GCG,FASTA, and ROBUST programs for amino acid sequence analysis, and theSIM, GAP, NAP, LAP2, GAP2, and PIPMAKER programs for nucleotidesequences. Preferred software analysis programs for both amino acid andpolynucleotide sequence analysis include the ALIGN, CLUSTALW (e.g.,version 1.6 and later versions thereof), and BLAST programs (e.g., BLAST2.1, BL2SEQ, and later versions thereof).

“Identity” (sometimes referred to as“overall identity”—in contrast to“local identity,” which is discussed further herein) with respect toamino acid or nucleotide sequences refers to the percentage of aminoacid residues or nucleotide bases, respectively, that are identical inthe two amino acid or nucleotide sequences when two such amino acidsequences or two such nucleotide sequences are optimally aligned withone another. If, in the optimal alignment, a position in a firstsequence is occupied by the same amino acid residue or nucleotideresidue as the corresponding position in the second corresponding aminoacid or nucleotide sequence, the sequences exhibit identity with respectto that residue position. The level of identity between two sequences(or “percent sequence identity”) is measured as a ratio of the number ofidentical positions shared by the sequences with respect to the size ofthe sequences analyzed (i.e., percent sequence identity=(number ofidentical positions/total number of positions) times 100).

Also encompassed in the present disclosure are nucleic acids thathybridize specifically under low, medium, or high stringency conditionsto a probe of at least 100 nucleotides from a nucleic acid encoding theamino acid sequence of SEQ ID NO:2 or to the nucleic acid sequence ofSEQ ID NOS:3 or 5.

Low stringency hybridization conditions, as used herein, include, e.g.,hybridization with a 100 nucleotide probe of about 40% to about 70% GCcontent; at 42° C. in 2×SSC and 0.1% SDS. Medium stringencyhybridization conditions include, e.g., at 50° C. in 0.5×SSC and 0.1%SDS. High stringency hybridization conditions include, e.g.,hybridization with the above-mentioned probe at 65° C. in 0.2×SSC and0.1% SDS. Under these conditions, as the hybridization temperature iselevated, a nucleic acid with a higher sequence homology is obtained.

Compositions Comprising eHLA-G Genetically Modified Cells

Also encompassed herein are pharmaceutical compositions, topicalcompositions, cellular grafts, and artificial tissues comprising orgenerated using one or more HLA-G modified mammalian cell types. Asshown herein, eHLA-G modified hESCs displayed stable and persistentHLA-G expression, even through directed differentiation into hEEPs.Furthermore, the stable and persistent HLA-G expression provided thegenetically modified cells with reduced immunogenicity and/or improvedimmunosuppression. In addition, it is shown herein that eHLA-G modifiedhuman dermal fibroblasts, which are fully differentiated cells, alsohave stable and persistent HLA-G expression that provides reducedimmunogenicity and/or improved immunosuppression. Thus, the eHLA-Gconstructs described herein can be used to generate universal donorcells of any type, whether from directed differentiation of agenetically modified pluripotent or multipotent cell, or from geneticmodification of a fully differentiated cell.

In one aspect, a topical composition for skin regeneration or repair isprovided that comprises a genetically modified dermal fibroblast cellcomprising an eHLA-G transgene as described herein. In another aspect, apharmaceutical composition for injection is provided that comprises agenetically modified dermal fibroblast cell comprising an eHLA-Gtransgene as described herein. In another aspect, a skin graftcomposition is provided that comprises a genetically modified dermalfibroblast cell comprising an eHLA-G transgene as described herein. Inanother aspect, a permanent skin graft composition is provided thatcomprises a genetically modified embryonic epidermal progenitor cellcomprising an eHLA-G transgene as described herein.

In another aspect, biocompatible synthetic scaffolds for artificialtissue and methods for their generation are described in the art and maybe used with the HLA-G modified cells described herein to produce anartificial tissue having reduced immunogenicity and/or improvedimmunosuppression as compared to tissues containing cells that do notexpress exogenous HLA-G. See, e.g., U.S. Pat. No. 7,960,166 entitled“Microfabricated compositions and processes for engineering tissuescontaining multiple cell types.”

II. Methods Cell Therapy Treatment

Because cells modified to stably express exogenous HLA-G in the mannerdescribed herein have reduced immunogenicity and/or increasedimmunosuppression, these traits allow the modified cell to serve as auniversal or improved donor cell or tissue. This is because the HLA-Gmediated reduction of immunogenicity and/or improvement inimmunosuppression provided to the cell can reduce or eliminate therequirement of matching the type of classical human leukocyte antigen(HLA) class I and class II molecules between donor cells and therecipient) for numerous injuries, diseases, or disorders.

Thus, HLA-G modified cells that stably express eHLA-G (and optionally inaddition, exogenous human β2 microglobulin), as described herein, may beused as for therapy. The therapy may be directed at treating the causeof the disease; or alternatively, the therapy may be to treat theeffects of the disease or condition. The genetically modified cells maybe transferred to, or close to, an injured site in a subject; or thecells can be introduced to the subject in a manner allowing the cells tomigrate, or home, to the injured site. The transferred cells mayadvantageously replace the damaged or injured cells and allowimprovement in the overall condition of the subject. In some instances,the transferred cells may stimulate tissue regeneration or repair,including skin regeneration or skin repair.

In various embodiments, HLA-G modified mammalian cells (e.g., humanHLA-G modified cells) are administered to a subject suffering from anyof a number of conditions including, but not limited to cardiovasculardisease, eye disease (e.g., macular degeneration), auditory disease,(e.g., deafness), diabetes, neurodegenerative disease, Alzheimer'sDisease, Parkinson's Disease, multiple sclerosis, osteoporosis, liverdisease, kidney disease, autoimmune disease, arthritis, gum disease, adental condition, or a proliferative disorder (e.g., a cancer). In othercases, the subject is suffering from, or at high risk of suffering from,an acute health condition, e.g., stroke, spinal cord injury, burn, or awound. In other cases, the subject is suffering from loss of tissue suchas lipatrophy or aging-related losses in collagen. In other cases, thesubject suffers from a non-healing ulcer, or is in need for an agent toassist in closure of defects like hypospadias and epispadias. In othercases, the subject is need for a permanent or temporary skin graft forwound healing or for skin substitutes.

In one aspect, the invention provides a universal method of cellular ortissue grafting to a subject in need thereof, the method comprisinginjecting or grafting to the subject a cellular or tissue compositioncomprising a population of eHLA-G modified cells, wherein the subjecthas at least one mismatched classical HLA class I or HLA class IImolecule as compared to the population of eHLA-G modified cells, andwherein the population of eHLA-G modified cells exhibits reducedimmunogenicity and/or improved immunosuppression as compared to cells ofthe same-type without the eHLA-G modification. The reducedimmunogenicity and/or improved immunosuppression can be determined, forexample, by comparing the eHLA-G modified cell to a control cell of thesame type without the eHLA-G modification in an NK-92 cytotoxicityassay, a humanized NSG tumor growth assay, and/or a PBMC proliferationassay.

In another aspect, the invention provides a method for regenerating skinto a subject in need thereof, the method comprising injecting apopulation of eHLA-G modified dermal fibroblasts and/or eHLA-G modifiedembryonic epidermal progenitors to a site of skin injury on the subject,wherein the subject has at least one mismatched classical HLA class I orHLA class II molecule as compared to the population of eHLA-G modifieddermal fibroblasts and/or eHLA-G modified embryonic epidermalprogenitors.

HLA-G modified cell types to be administered to a subject in needthereof include, but are not limited to, epidermal progenitor cells,mesenchymal stem cells, pancreatic β cell progenitors, pancreatic βcells, cardiac progenitors, cardiomyocytes, hepatic progenitors,hepatocytes, muscle cell progenitors, muscle cells, kidney cells,osteoblasts, hematopoietic progenitors, dental follicle cells, hairfollicle cells, retinal pigment epithelial cells, neural stem cells,neurons, astrocytes, oligodendrocytes, or any combination thereof. Suchmammalian cells can be derived from one of several species including,e.g., human, mouse, rat, monkey, or pig.

The therapy may be directed at treating the cause of the disease; oralternatively, the therapy may be to treat the effects of the disease orcondition. The HLA-G modified cells may be transferred to, or close to,an injured site in a subject; or the cells can be introduced to thesubject in a manner allowing the cells to migrate, or home, to theinjured site. The transferred cells may advantageously replace thedamaged or injured cells and allow improvement in the overall conditionof the subject. In some instances, the transferred cells may stimulatetissue regeneration or repair.

The transferred cells may be cells differentiated from HLA-G modifiedpluripotent (or totipotent) stem cells. The transferred cells also maybe multipotent stem cells differentiated from pluripotent, HLA-Gmodified cells.

The number of administrations of treatment to a subject may vary.Introducing the HLA-G modified and/or differentiated cells into thesubject may be a one-time event; but in certain situations, suchtreatment may elicit improvement for a limited period of time andrequire an on-going series of repeated treatments. In other situations,multiple administrations of the cells may be required before an effectis observed. As will be appreciated by those of ordinary skill in theart, the exact treatment protocols will depend upon the disease orcondition, and the stage of the disease and parameters of the individualsubject being treated.

The HLA-G modified cells may be introduced to the subject via any of thefollowing routes: parenteral, intravenous, intraarterial, intramuscular,subcutaneous, transdermal, intratracheal, intraperitoneal, or intospinal fluid.

The HLA-G modified cells may be differentiated into cells and thentransferred to subjects suffering from a wide range of diseases ordisorders.

Pancreatic islet cells (or primary cells of the islets of Langerhans)may be transplanted into a subject suffering from diabetes (e.g.,diabetes mellitus, type 1), see e.g., Burns et al., (2006) Curr. StemCell Res. Ther., 2:255-266. Thus, in some embodiments, pancreatic betacells derived from HLA-G modified cells are transplanted into a subjectsuffering from diabetes (e.g., diabetes mellitus, type 1).

In other examples, hepatic cells or hepatic stem cells derived fromHLA-G modified cells are transplanted into a subject suffering from aliver disease, e.g., hepatitis, cirrhosis, or liver failure.

Degenerative heart diseases such as ischemic cardiomyopathy, conductiondisease, and congenital defects could benefit from stem cell therapies.See, e.g., Janssens et al., (2006), Lancet, 367:113-121.

Hematopoietic cells or hematopoietic stem cells (HSCs) derived fromHLA-G modified cells may be transplanted into a subject suffering fromcancer of the blood, or other blood or immune disorder. Examples ofcancers of the blood that are potentially treated by hematopoietic cellsor HSCs include: acute lymphoblastic leukemia, acute myeloblasticleukemia, chronic myelogenous leukemia (CML), Hodgkin's disease,multiple myeloma, and non-Hodgkin's lymphoma. Often, a subject sufferingfrom such disease must undergo radiation and/or chemotherapeutictreatment in order to kill rapidly dividing blood cells. IntroducingHSCs derived from HLA-G modified cells to these subjects may help torepopulate depleted reservoirs of cells.

Subjects suffering from neurological diseases or disorders couldespecially benefit from HLA-G modified cell therapy, especially when theblood/brain barrier may have been compromised. In some approaches, theHLA-G modified cells may be differentiated into neural stem cells orneurons and then transplanted to an injured site to treat a neurologicalcondition, e.g., Alzheimer's disease, Parkinson's disease, multiplesclerosis, cerebral infarction, spinal cord injury, or other centralnervous system disorder, see, e.g., Morizane et al., (2008), Cell TissueRes., 331(1):323-326; Coutts and Keirstead (2008), Exp. Neurol.,209(2):368-377; Goswami and Rao (2007), Drugs, 10(10):713-719.

For the treatment of Parkinson's disease, the HLA-G modified cells maybe differentiated into dopamine-acting neurons and then transplantedinto the striate body of a subject with Parkinson's disease. For thetreatment of multiple sclerosis, neural stem cells may be differentiatedinto oligodendrocytes or progenitors of oligodendrocytes, which are thentransferred to a subject suffering from MS.

For the treatment of any neurologic disease or disorder, a successfulapproach may be to introduce neural stem cells to the subject. Forexample, in order to treat Alzheimer's disease, cerebral infarction or aspinal injury, the HLA-G modified cells may be differentiated intoneural stem cells followed by transplantation into the injured site. TheHLA-G modified cells may also be engineered to respond to cues that cantarget their migration into lesions for brain and spinal cord repair,e.g., Chen et al., (2007), Stem Cell Rev., 3(4):280-288.

Optionally, the HLA-G modified cells to be used in cell therapy methodsalso express a reporter protein as described herein. In someembodiments, the reporter protein to be used is one that facilitates invivo detection (e.g., imaging) of the introduced cells. For example, thecells may express a far-red emitting fluorescent protein such asKatushka, whose long excitation and emission wavelengths are well suitedto imaging in tissues. Katushka is commercially available under thetradename “TurboFP635” (Evrogen, Moscow, Russia).

EXAMPLES

The following specific examples are to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever. Without further elaboration, it is believed that oneskilled in the art can, based on the description herein, utilize thepresent invention to its fullest extent. All publications cited hereinare hereby incorporated by reference in their entirety. Where referenceis made to a URL or other such identifier or address, it is understoodthat such identifiers can change and particular information on theinternet can come and go, but equivalent information can be found bysearching the internet. Reference thereto evidences the availability andpublic dissemination of such information.

Example 1 Identification of Gene Expression Patterns Associated withImmune Tolerance of Cancer Cells

Human soft tissue cancer arrays were initially screened in search of acandidate immune tolerance gene that showed increased expression withcancer progression to metastatic states, presumably due to evasion ofimmune surveillance mechanisms. The data revealed a strong positivecorrelation between successful metastasis and expression levels ofseveral genes previously implicated in the immune tolerance. To assesswhether MSCs could induce immune tolerance and allograft acceptance,MSCs were cross-screened by RT-PCR to examine the expression levels ofthese candidate genes and several antigenic HLAs. Table 1 illustratesthat passage 1 MSCs expressed HLA class Ia, HLA-G and II markers inaddition to CD200, CD47, and indoleamine 2,3-dioxygenase (IDO). It wasfound that a population of MSCs expressed HLA-G at moderate levels,albeit less than that found for Jeg-3, a cancer line with aggressivemetastatic potential.

TABLE 1 Passage HLA HLA Cell type # class Ia HLA-G class II IDO CD200CD47 MSC G⁻ 1 ++++ − −/+ + ++ + 3 ++++ − ++ + ++ + MSC G⁺ 1 ++++ ++−/+ + ++ + 3 ++++ − ++ + ++ + Jeg3 1 ++ +++ −/+ − − + 3 ++ +++ −/+ − − +

Since the immunosuppressive effect of MSCs in vivo appears to betransient, MSCs were serially passaged and monitored for changes in theexpression levels of the selected genes. By passage 3, native HLA-Gexpression was absent in MSCs, although other candidate markers remainedunchanged. HLA-G expression was not altered in Jeg-3 cells. MSCs werethen isolated based on expression of cell surface HLA-G using FACS andfound that between 0.5-3% of MSCs expressed HLA-G at the cell surface.To assess the ability of HLA-G⁺ MSCs to escape donor rejection in vivo,1-3×10⁵ cells were injected into the tail veins of immunocompetent mice.Blood samples (200 μl) were collected from the retro-orbital plexus andFACS sorted using an anti-human HLA class I specific mAb. Table 2 showsthat 1 week post-transplantation, HLA-G+ MSCs (and Jeg-3 cells)demonstrated a 24- and 270-fold survival advantage over HSCs and Jurkatcells (both HLA-G⁻), respectively. By two weeks, the advantage of HLA-G⁺MSCs increased to 27-and 311-fold. HLA-G⁻ MSCs survived at the same rateas HSCs, suggesting that the HLA-G⁺ subpopulation of MSCs may exhibitenhanced tolerizing effects relative to unsorted MSCs in an in vivosetting. Indeed, post-mortem analysis revealed frank tumors in the lungsof immunocompetent mice 12 weeks post-transplantation with Jeg-3 cells,but not G⁺ MSCs (data not shown).

TABLE 2 Cell Type HLA-G Sampling Time % Survival 100k MSCs − 2 weeks0.51% 100k MSCs + 1 week 10.82%  2 weeks 12.47%  150k Jeg-3 + 1 week13.5% 300k HSCs − 1 week 0.46% 300k Jurkat − 1 week 0.04%

Next, a modified HLA-G construct was overexpressed in HF (humanfibroblast) and K562 cells and tested for protection against lysis byhuman NK cells (Table 3). NK-mediated cytotoxicity was reduced by 75% inHLA-G⁺ HF and virtually eliminated in HLA-G+ K562 cells. This isconsistent with the observed protection of HLA-G⁺ Jeg-3, which wasreversed by incubation with the neutralizing anti-HLA-G (87G) antibodybut not an isotype control. These data suggest that protection from NKkilling was HLA-G-dependent.

TABLE 3 HLA-G Surface Cytotoxic Promoter ER Retrieval 3′ UTR Exp Lysis(3:1) Silencing pMSCV wildtype absent low- 32-41% 4-6 weeks medium pMSCVwildtype wildtype low 40-60% 4-6 weeks pMSCV mutated wildtype medium25-33% 4-6 weeks pMSCV^(mut) wildtype absent low- 35-42% None @ medium12 mo pMSCV^(mut) mutated mutated high 0-8% None @ 5 mo

Previous studies showed that the mutated MSCV promoter, M-U3/R, avoidedsilencing pressure through 10 weeks of culture. Our studies showed thatM-U3/R resisted silencing after 1 year of continuous culture, superiorto the wildtype promoter which was silenced by 4-6 weeks of culture.Moreover, mutation or deletion of the 3′ UTR enhanced HLA-G surfaceexpression, as did mutation of the ER retrieval motif. Higher HLA-Gsurface expression negatively correlated with cytotoxic lysis,reinforcing the importance of employing an optimal gene deliveryconstruct.

Example 2 Culture and Differentiation of Human Embryonic Stem Cells intoHuman Epidermal Progenitors (hEEPs)

All tissue culture reagents were from Life Technologies unless otherwisespecified. ESC growth medium contains DMEM/F12 (1:1) supplemented with20% knockout serum replacement, 0.1 mM MEM non-essential amino acids, 1mM GlutaMax, 0.1 mM β-mercaptoethanol (Sigma). ESC growth medium wasconditioned by plating mitotically inactivated mouse embryonicfibroblasts (MEFs) (CF-1, ATCC) at a density of 5×10⁴ cells/cm² andincubating for 18-24 hours. After conditioning, 4 ng/ml bFGF was addedand complete conditioned medium was sterile filtered. hESCs weresubcultivated every 5-6 days (1:3 or 1:4 split) on Matrigel-coatedplates using 1 mg/ml Dispase to remove cell colonies. K14⁺/p63⁺ hEEPswere generated according to the method of Metallo et al supra. Briefly,hESCs were cultured in 6-well plates for 4 days and then treated with 2ml/well of differentiation medium, comprised of unconditioned hESCgrowth medium containing 1 μM all-trans retinoic acid (Sigma) and 25ng/ml BMP4. After daily medium changes for 7 days, cells were treatedwith dispase, centrifuged, and resuspended in defined keratinocyteserum-free medium (DFSM) and seeded on gelatin-coated plates at a splitratio of 1:3. DSFM was changed every other day for 3-4 weeks. Cells werethen subcultured by trypsinization, centrifuged, washed, and plated at10,000 cells per cm² on gelatin-coated tissue culture plates in DSFM.After 14 days in defined keratinocyte serum-free medium, early signs ofepidermal differentiation was observed by microscopy as characterized bythe formation of an epidermal sheet structure. After four weeks ofculturing, cells in epidermal sheets displayed typical epidermaldifferentiation phenotype with cubic morphology.

Isolation of total RNA from cells and reverse transcriptase reactionswere described previously in Zhao et al (2010), Tissue Eng Part A,16(2):725-733. Specific PCR amplification was performed in the HybaidOmnigene thermal cycler (Bio-rad, Hercules, Calif.) using specificprimers of the genes of interest as shown in FIG. 12. PCR conditionsconsisted of 35 cycles at 94° C. for 30 s, 65° C. for 1 min, and 72° C.for 1 min with a final extension at 72° C. for 10 min. Ten pi of eachPCR product was detected by ethidium bromide gel electrophoresis. TheFIG. 12 data indicates that the epidermal differentiation markers K14,Tap63, and ΔNp63 were gradually enhanced during differentiation. In datanot shown here, immunofluorescence studies of K14 and additionalepidermal markers p63, CD29, and CD49f were conducted. DifferentiatedeHLA-G(EF-1α)-GFP hEEPs were positive for K14, p63, CD29, and CD49fprotein expression as indicated by immunofluorescence.

To verify that epithelial monolayers were of ≧90% purity and expressK14, cells were subjected to flow cytometry according to the method ofMetallo et al supra, and analyzed on a BD FACS Canto II. The impact ofeHLA-G transgene expression (eHLA-G(EF1-α)-GFP-hESCs) on hESCdifferentiation into EPs (epidermal progenitors) was assessed bycomparing the degree of K14 positivity for wildtype, G⁻, and G⁺-hEEPs.To enhance purity of isolated hEEPs, cells were sorted using magneticactivated cell sorting (MACS) with CD29 antibodies. About 92 percent ofCD29 MACS sorted hEEP cell culture cells differentiated fromeHLA-G(EF-1α)-GFP modified hESCs were positive for K14, a specifickeratinocyte marker. Purified hEEPs exhibited homologous keratinocytemorphology as shown by phase contrast microscopy (see FIG. 16).

The stability of the HLA-G transgene in differentiated hEEPs wasconfirmed by flow cytometry. Both HLA-G total expression and surfaceexpression was robust for differentiated eHLA-G(EF-1α)-GFP-hEEPs(greater than 90% of cells) as compared to control cells with noexogenous HLA-G (GFP only hEEPs) and wild-type hEEPs (see FIG. 17). Onlyclones that yield a similar differentiation potential as wildtype cellswere selected for further study.

Example 3 eHLA-G Construct Design and Stable Expression in hESCs

A novel HLA-G construct was designed by combining multiplemodifications: 1) mutation of HLA-G's ER retrieval motif (K334A/K335A);and 2) mutation of HLA-G's 3′ UTR microRNA binding sites. Since viralgene delivery systems remain a serious regulatory challenge, thePiggyBac system was used, a transposon-based, nonviral approach that wasrecently shown to achieve a 90% transfection efficiency in H1 hESCs(Lacoste et al (2009), Cell Stem Cell, 5:332-342.). This system requiresa donor plasmid containing the transposon (FIG. 1A) and a helper plasmidexpressing the transposase (FIG. 2). To generate helper plasmids, theePiggyBac codon humanized transposase cDNA was custom synthesized(GeneArt) and then cloned in pBluescript (Stratagene) downstream of aPGK promoter and upstream of an SV40 polyadenylation signal sequence(pA). For the eHLA-G expression cassette, multiple promoters werecompared including the M-U3/R promoter, MSCV promoter, and the Chinesehamster EF1α (CHEF-1α) promoter, the sequence of which is provided belowas SEQ ID NO:6.

one embodiment of the CHEF-1α promoter (SEQ ID NO: 6) GGATGGCGGGGCTGACGTCGGGAGGTGGCCTCCACGGGAAGGGACACCCGGATCTCGACACAGCCTTGGCAGTGGAGTCAGGAAGGGTAGGACAGATTCTGGACGCCCTCTTGGCCAGTCCTCACCGCCCCACCCCCGATGGAGCCGAGAGTAATTCATACAAAAGGAGGGATCGCCTTCGCCCCTGGGAATCCCAGGGACCGTCGCTAAATTCTGGCCGGCCTCCCAGCCCGGAACCGCTGTGCCCGCCCAGCGCGGCGGGAGGAGCCTGCGCCTAGGGCGGATCGCGGGTCGGCGGGAGAGCACAAGCCCACAGTCCCCGGCGGTGGGGGAGGGGCGCGCTGAGCGGGGGCCCGGGAGCCAGCGCGGGGCAAACTGGGAAAGTGGTGTCGTGTGCTGGCTCCGCCCTCTTCCCGAGGGTGGGGGAGAACGGTATAAAAGTGCGGTAGTCGCGTTGGACGTTCTTTTTCGCAACGGGTTTGCCGTCAGAACGCAGGTGAGTGGCGGGTGTGGCCTCCGCGGGCCCGGGCTCCCTCCTTTGAGCGGGGTCGGACCGCCGTGCGGGTGTCGTCGGCCGGGCTTCTCTGCGAGCGTTCCCGCCCTGGATGGCGGGCTGTGCGGGAGGGCGAGGGGGGGAGGCCTGGCGGCGGCCCCGGAGCCTCGCCTCGTGTCGGGCGTGAGGCCTAGCGTGGCTTCCGCCCCGCCGCGTGCCACCGCGGCCGCGCTTTGCTGTCTGCCCGGCTGCCCTCGATTGCCTGCCCGCGGCCCGGGCCAACAAAGGGAGGGCGTGGAGCTGGCTGGTAGGGAGCCCCGTAGTCCGCATGTCGGGCAGGGAGAGCGGCAGCAGTCGGGGGGGGGACCGGGCCCGCCCGTCCCGCAGCACATGTCCGACGCCGCCTGGACGGGTAGCGGCCTGTGTCCTGATAAGGCGGCCGGGCGGTGGGTTTTAGATGCCGGGTTCAGGTGGCCCCGGGTCCCGGCCCGGTCTGGCCAGTACCCCGTAGTGGCTTAGCTCCGAGGAGGGCGAGCCCGCCCGCCCGGCACCAGTTGCGTGCGCGGAAAGATGGCCGCTCCCGGGCCCTGTAGCAAGGAGCTCAAAATGGAGGACGCGGCAGCCCGGCGGAGCGGGGCGGGTGAGTCACCCACACAAAGGAAGAGGGCCTTGCCCCTCGCCGGCCGCTGCTTCCTGTGACCCCGTGGTGTACCGGCCGCACTTCAGTCACCCCGGGCGCTCTTTCGGAGCACCGCTGGCCTCCGCTGGGGGAGGGGATCTGTCTAATGGCGTTGGAGTTTGCTCACATTTGGTGGGTGGAGACTGTAGCCAGGCCAGCCTGGCCATGGAAGTAATTCTTGGAATTTGCCCATTTTGAGTTTGGAGCGAAGCTGATTGACAAAGCTGCTTAGCCGTTCAAAGGTATTCTTCGAACTTTTTTTTTAAGGTGTTGTGAAA ACCACCG

To generate the donor plasmid, T53C/C136T mutant 5′ terminal repeat (TR)of 313 bp and 3′ TR of 235 bp (as described in Lacoste supra) werecustom synthesized and cloned upstream and downstream, respectively, ofthe following expression cassette: eHLA-G, a 250 bp chicken β-globin HS4core insulator (Recillas-Targa et al (2002), PNAS USA, 99:6883-6888),EGFP, and pA. HS4 was used to prevent spreading of repressive chromatininto the integrated construct. EGFP expression was driven by thephosphoglycerate kinase (PGK) promoter (see FIG. 1A). The sequence ofthe HS4 element is shown below as SEQ ID NO:7.

one embodiment of the HS4 element (SEQ ID NO: 7)GAGCTCACGGGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGATACGGGGAAAAAGCTT

The empty vector donor plasmid (HLA-G⁻) was identical to the eHLA-Gdonor plasmid except that the eHLA-G construct was excluded (FIG. 1B).Prior to gene transfer, hESCs were treated for 1 hr with 10 μM Y-27632,a ROCK inhibitor shown to substantially reduce dissociation-inducedapoptosis and increases cloning efficiency (Watanabe et al (2007), NatBiotechnol, 25:681-686. hESCs were dissociated in 0.25% trypsin-EDTA at37° C. for five minutes, washed in conditioned mTeSR medium plusY-27632, and resuspended in nucleofection solution L (Amaxa). 3 μg ofhelper and 6 μg of transposon donor plasmids were added per 1.5×10⁵cells, and nucleofection was performed with program setting B-016. hESCswere then plated in CM plus Y-27632 at 2×10⁵ cells per 6 cm dish forclonal selection. After 24 hours, the culture medium was changed to CMalone, then changed daily thereafter. Clones with the highest dualexpression of tdT/eHLA-G were selected using fluorescence microscopyrather than via antibiotic resistance or flow cytometry since transgenesilencing is frequent in hESCs and only a fraction of single transgeniccells gives rise to a marked cell line (Braam et al (2008), Nat Methods,5:389-392).

Example 4 Assessment of the ePiggyBac Gene Delivery System

eHLA-G transfection efficiency was determined by plating the transfectedcells in CM plus Y-27632 at 2×10³ cells per 6 cm dish for 24 hours, andthen changing to CM alone. Medium was then changed daily for seven days,and colonies were evaluated by live cell staining and immunofluorescencemicroscopy, as discussed in Example 5. For each clone, three high powerfields were counted and the percentage of reporter protein⁺/eHLA-G⁺hESCs was calculated. Results of this experiment are shown in FIGS. 3-4.The eHLA-G insertion site was determined using a plasmid rescue strategyas described in Lacoste et al supra. Briefly, genomic DNA was isolatedfrom transgenic hESC clones and digested with BamHI/BgIII/NotI,self-ligated at low concentration with T4 DNA ligase overnight at 16°C., precipitated with 100% isopropanol, and washed with 70% ethanolbefore transformation in DH10B E. coli and selected on ampicillin.eHLA-G copy number was determined using SplinkTA PCR. Standard G-bandingwas performed every 20 passages to assess karyotype stability. eHLA-Gand reporter protein gene silencing were assessed every 10 passagesusing flow cytometry. Prior to dissociation and analysis, hESCs weretreated for one hour with 10 μM Y-27632. Cells were then dissociatedwith 0.25% trypsin-EDTA at 37° C. for five minutes, washed in CM plusY-27632, resuspended in ice-cold PBS containing 0.1% BSA and 0.5 mMEDTA, and then analyzed using a BD FACS Canto II flow cytometer (BectonDickinson). As shown in FIGS. 8 and 9, at passage 16, essentially nosilencing of EGFP or eHLA-G expression was observed. Pluripotency wasassessed every 20 passages by immunocytochemical detection of thefollowing: 1) pluripotency markers Oct3/4, SSEA-4, Sox2 and Nanog (FIGS.5 and 6), 2) reporter protein⁺/eHLA-G⁺ embryoid body formation (FIG. 7),and 3) the endodermal marker Gata 6, mesodermal marker muscle actin, andectodermal marker neurofilament heavy chain in differentiated transgenichESCs (data not shown), all using a Leica CTR6500 fluorescentmicroscope. All antibodies were from Abcam unless otherwise specified.

Example 5 Cell Surface Localization of eHLA-G and Other HLA Proteins

Live cell staining was used to detect cell surface HLA class Ia, HLA-E,eHLA-G, and HLA class II expression in transgenic versus wildtype hESCsand hEEPs. Briefly, cells were harvested and washed in cold PBS, stainedwith the corresponding 1 mAb in PBS containing 10% goat serum and 3% BSAfor 60 min at 4° C., washed, fixed with 1% paraformaldehyde for 10 min,and subsequently stained with a goat anti-mouse IgG conjugated with FITCfor 30 minutes at 4° C. Control aliquots were stained with anisotype-matched IgG to evaluate nonspecific binding to target cells.Each antibody (MEMG/9 for HLA-G, MEM-E/08 for HLA-E, Bub for HLA classIa, and HKB1 (Abbiotec, San Diego, Calif.) for HLA class II) was firsttested at several dilutions in order to determine the optimal conditionsfor achieving specific-only binding. After staining, cells were smearedon a glass slide, allowed to air dry, and then mounted with anti-fademedia containing DAPI (Vector Laboratories). Slides were observedimmediately under a Leica CTR6500 fluorescence microscope. As shown inFIGS. 5 and 10, expression of HLA-A,B,C; HLA-E, HLA-DP, DQ, DR, andβ-microglobulin were similar in wildtype and eHLA-G modified human EScells, whereas an approximately seven-fold higher level of HLA-Gexpression was observed in the eHLA-G modified human ES cells.

Example 6 Assessment of eHLA-G Expression on NK-92 Cell-InducedCytotoxicity

eHLA-G-expressing hESCs were cultured and differentiated into hEEPs asdescribed in Example 2. NK-92 cells (CRL-2407, American Type CultureCollection, Manassas, Va.) were cultured in Minimum Essential MediumAlpha Medium (α-MEM, Invitrogen) supplemented with 12.5% FBS, 12.5%horse serum, 0.2 mM inositol, 0.1 mM β-mercaptoethanol, 0.02 mM folicacid and 100 IU/ml recombinant IL-2 (Sigma) at 37° C. in a 5% CO₂humidified incubator.

Cytotoxicity was performed using a CytoTox96 Non-RadioactiveCytotoxicity Assay Kit (Promega, Madison, Wis.) as the protocolinstructed. Briefly, effector cells were mixed with 5×10³ target cellsat various NK-92 (Effector or “E”) to hESCs or hEEPs (target or “T”) E:Tcell ratios in U-bottom 96 well plates (Costar, Cambridge, Mass.). After4 h at 37° C. in a humidified 5% CO₂ incubator, 50 μl of the supernatantwas collected to determine the LDH release. Target cell spontaneousrelease and maximal release of LDH and the effector cell spontaneousrelease of LDH were determined by incubating these cells in mediumalone. Each assay was performed in triplicate and the results wereexpressed as percentages of lysis %. The percentage of specific lysiswas determined as follows: (experimental release−effector spontaneousrelease−target spontaneous release/target maximum release−targetspontaneous release)×100. In all experiments spontaneous release was<10% of maximum release.

As shown in FIG. 11, at an E:T ratio of 1:10, killing of eHLA-Gexpressing hESCs was reduced by over 50% relative to wildtype cellsexpressing GFP alone. At an E:T of 1:30, killing of eHLA-G expressinghESCs was reduced by approximately 75%. Wildtype hESCs were killed at areasonable rate as shown for both E:T ratios (GFP alone). Further, itwas shown that expression of the 3′ UTR (Del 14 bp) HLA-G allele in K562cells results in diminished NK cell-induced cytotoxicity relative tothat observed in K562 cells expressing the (Ins 14 bp) HLA-G allele, aswell as in unmodified K562 cells. See FIG. 14.

The results of FIG. 11 were repeated and confirmed in additional NKcytotoxicity experiments. As shown in FIG. 18, killing ofeHLA-G(EF-1α)-GFP-hESCs was reduced more than 100% as compared tocontrols hESCs that contained only a GFP transgene (no HLA-G transgene).This data shows that HLA-G transgene expression impartsimmunosuppressive and/or reduced immunogenicity characterstics in hESCs.

NK cytotoxicity experiments were also conducted on hEEPs differentiatedfrom hESCs. As shown in FIG. 19, killing of eHLA(EF-1α)-GFP-hEEPsdifferentiated from eHLA(EF-1α)-GFP-hESCs was reduced well more than100% (about 3-fold) as compared to control hEEPs. This data shows thateHLA(EF-1α)-GFP transgene is stable and persistent throughout theprocess of differentiation, and that HLA-G expression is able to impartimmunosuppressive and/or reduced immunogenicity characteristics indifferentiated cells.

Example 7 Determination of the Immunogenicity of e-HLA-G⁺ Cells in Vivoby Allografts in Humanized Mice

A humanized mouse model with human peripheral blood lymphocytes(Hu-PBL-NSG), but not wildtype immunodeficient NSG mice, was recentlyshown to reject mismatched human islets within 1-2 weekspost-transplantation (King et al (2008), Clin Immunol, 126:303-314).Although graft-versus-host-disease (GVHD) sets in at 4-5 weeks, graftsurvival is monitored until euthanasia criteria are met. The presence ofonly lower levels of GVHD allows us to extend our observation window.NSG mice (females at six weeks of age) were purchased from JacksonLaboratory and handled in accordance with the guidelines of theInstitutional Animal Care and Use Committee and the recommendations inthe Guide for the Care and Use of Laboratory Animals (Institute ofLaboratory Animal Resources, National Research Council, National Academyof Sciences). Functional humanized NSG mice were generated byintravenous injection of about 20×10⁶ human PBMCs into NSG miceaccording to (Pearson et al (2008), Curr Protoc Immunol, Ch.15:Unit15.21; and King et al supra). Engraftment was verified at aboutfour weeks by collecting blood from the retroorbital venous plexus ofanesthetized mice using EDTA-coated capillary tubes (DrummondScientific) and EDTA-treated 1.5 ml tubes (Eppendorf). Cells were thenprocessed for human CD45 positivity by FACS analysis according to Kinget al supra. Levels of human CD45⁻ cells reaching 0.1% in the blood atfour weeks is considered a successful engraftment and allowsallorejection studies.

The hESC and hEEP culture systems that are typically used, expose cellsto immunogenic animal derivatives such as the sialic acid Neu5Gc (Martinet al (2005), Nat Med, 11:228-232). Thus, a decontamination step can beadded that was recently shown to significantly reduce Neu5Gc levels(Heiskanen et al (2007), Stem Cells, 25:197-202). hESCs can bedecontaminated of Neu5Gc using two approaches: 1) culturing in TeSR1media supplemented with bFGF, LiCl, GABA, and pipecolic acid on humanmatrix coated plates according to the method of Ludwig et al (2006), NatBiotechnol, 24:185-187, and 2) KSR replacement by heat-inactivated bloodgroup AB Rh⁻ human serum (Heiskanen et al (2007), Stem Cells,25:197-202. hESCs cultured under both methods can be assessed byincubation with anti-Neu5Gc mAb and analyzed by flow cytometry. Thesecond approach may be adopted, as the method of Ludwig et al mayrequire hESC adaptation. hEEPs are cultured in DFSM that lacks animalderivatives per its manufacturer.

The objective of performing allografts with HLA-G modified cells is toassess whether HLA-G expression reduces immunogenicity as indicated byan increase in tumor size. Prior to transplantation, the intendedinjection site was shaved to facilitate clinical observation. Animalguidelines including appropriate use of anesthesia were followed at alltimes. 5×10⁶ transgenic eHLA-G⁺ and HLA-G⁻ hESCs were resuspended in 100μl of the appropriate media containing India ink to mark the injectionsite. This facilitates histological assessment should insufficientfluorescence be observed. Cells were injected subcutaneously into thethoracic mammary fat pad of five 3-month old Hu-PBL-NSG mice. Theresults of these allografts are shown in FIGS. 20 and 21. The “G0” hESCsare the control ESCs that do not contain an eHLA-G transgene, but ratheronly GFP. “mG1(#1)” and “mG1(#2)” refer to two differenteHLA-G(EF-1α)-GFP nucleofected hESC clones. The G0, mG1(#1), and mG1(#2)tumors, as shown in FIG. 20, were measured and weighed. The G0 hESCsformed a tumor with a volume of 126.9 cubic millimeters and a weight of32 milligrams. The mG1(#1) hESCs formed a tumor with a volume of 748.4cubic millimeters and a weight of 318 milligrams. The mG1(#2) hESCsformed a tumor with a volume of 1116.7 cubic millimeters and a weight of675 milligrams.

FIG. 21 shows the averaged results of tumors from hESC allografts ontofive humanized NSG mice. The data shows that HLA-G nucleofected hESCs(“mG1”) formed much larger (more than 3-fold by volume) and heavier(more than 2-fold by weight) tumors than wild-type hESCs (“G0”)transplanted into humanized NSG mice. Thus, the data indicates thateHLA-G transgene expression can provide reduced immunogenicity and/orincreased immunosuppression. This supports the general application ofthe eHLA-G transgene constructs described herein for modifying anydesired cell-type into a universal or superior allogenic donor fortherapy, transplants, tissue repair, cell and tissue substitutes, andthe like.

The above-described allograft experiments with humanized NSG mice can beconducted with any eHLA-G modified cell-type that actively proliferatesor can be induced to proliferate.

In addition, allografts in humanized mice can be monitored using aSpectrum In Vivo Imaging System (Caliper, Mountain View, Calif.) with a620 nm emission filter and 2.5 second exposure time. At wavelengths of620 nm or greater, the autofluorescence experienced with GFP iseliminated and signal can be detected as deep as 2.5 cm beneath thesurface of the skin (Shaner et al (2004), Nat Biotechnol, 22:1567-1572).Mice are longitudinally imaged and weighed on a weekly basis. If nosignal is observed during the four week period, mice are sacrificed andthe injected thoracic mammary fat pad is assessed histologically usingfluorescence microscopy to detect the fluorescent reporter protein used.In all cases, upon sacrifice of mice, tissue is stained with hematoxylinand eosin to assess for teratoma formation and immune rejection.Immunohistochemistry is also be performed to detect human β2microglobulin (PBMCs, hESCs, hEEPs) and CD45 (PBMCs), with the latterserving as a supplemental measure of immune cell infiltration of theallograft. Slides are read by an expert pathologist blinded to theexperimental conditions.

Example 8 Assessment of the Tumorigenicity of eHLA-G+- and HLA-G⁻-hEEPs

The impact of eHLA-G on hEEP tumorigenicity is assessed by injectingtransgenic G⁺ and non-transgenic G⁻-hEEPs using a similar protocol asdiscussed in Example 8 except that cells are transplanted intonon-humanized NSG mice. A total of 14 mice are monitored usinglongitudinal fluorescent live animal imaging and weight assessment on amonthly basis for up to the earlier of nine months or meeting euthanasiacriteria. Mice are then sacrificed and the injected thoracic mammarygland harvested in 4% neutral-buffered paraformaldehyde. Fixed tissue istransferred to 70% ethanol and embedded in paraffin. Sections arestained with hematoxylin and eosin and processed for fluorescentreporter protein microscopy. If the tissue sections are found to bereporter protein-negative, they are stained using mAbs specific to humanβ2 microglobulin and CD45, with the latter serving to ensure thepresence of non-PBMCs at the injection site.

The statistical significance of independent means is assumed for pvalues of <0.05. Two-sample mean comparisons are determined using aStudent's t test. Comparisons of three or more means are made using one-or two-way analyses of variance, and Bonferroni's post-hoc test. Allmeasures of variance are presented as standard error of the mean. Alinear regression is performed to compare the relationship between invitro and in vivo data to determine if the former has predictive valuetowards human allograft rejection in the Hu-PBL-NSG mouse model that isused in this study. For example, the percentage increase inalloproliferation in vitro will be regressed against the PBMC-matchedpair used in the in vivo phase (output is the level of reporter proteinfluorescence at four weeks post-transplantation). A successful outcomehere is a high R² value and negative slope coefficient, suggesting thateHLA-G expression leads to increased engraftment. Such an interpretationis contingent on a high correlation between eHLA-G expression andreporter protein positivity in the clones selected for use in the invitro and in vivo studies.

Example 9 Immunosuppression and Immunogenicity Assessment of eHLA-GStably Transfected into Fully Differentiated Fibroblasts

The eHLA-G(EF-1α)-GFP transgene and control constructs were transfectedinto human newborn dermal fibroblasts (a cell-type that is already fullydifferentiated) using nucleofection. Human newborn dermal fibroblastcells (HFD) were purchased from ATCC and cultured in Iscove's ModifiedDulbecco's Medium (IMDM) (ATCC) supplemented with 10% FBS and 1% PS(Invitrogen). When the cells reached 80% confluence, cells wereharvested by incubating with 0.25% Trypsin-EDTA (Invitrogen) for 3minute at 37° C. Cells were counted and 0.5×10⁶ were centrifuged andresuspended in human dermal fibroblast nucleofection solution (Cat. No.VPD-1001, Lonza, Walkersville, Md.). Helper and transposon plasmids wereadded to the cell suspension, and nucleofection was performed withprogram setting U-020 according to the manufacturer's protocol (Lonza).Cells were then plated in 6-well plates and incubated in humidified 37°C./5% CO₂ for 24 h. After 24 h, the stable transfected cells wereselected with 1 μg/ml puromycin (Sigma) for 7 days. Stable GFP-positivecells were maintained in 500 ng/ml puromycin and HLA-G and GFPexpression were detected with flow cytometry. Stable transfectants weremaintained in culture for use in peripheral blood mononuclear cell(PBMC) proliferation assays and NK-92 cytotoxicity assays as describedbelow.

PBMC proliferation assays. Human dermal fibroblasts stably transfectedwith the eHLA-G(EF-1α)-GFP transgene (“HFD-m1-GFP” cells) or GFP-alonecontrol construct (“HFD-G0-GFP” cells) were assessed for their abilityto inhibit PBMC proliferation. HFD-G0-GFP and -mG1-GFP cells wereinactivated with Mitomycin C (10 μg/ml for 2.5 h) and seeded at3.0×10³/well in 96-well plate and allowed to adhere for 24 h. 1×10⁵ PBMCin the presence of 6 μg/ml PHA were added in triplicates in thecorresponding HFD cells. HFD-G0-GFP and -mG1-GFP cells alone wereincluded for MTT-OD correction. (“MTT” is a pale yellow substratereagent that is cleaved by living cells to yield a dark blue formazanproduct. This process requires active mitochondria, and even freshlydead cells do not cleave significant amounts of MTT. MTT thereforeprovides a colorimetric assay that can be used for either proliferationor cytotoxicity assays.) PBMC without PHA and PBMC with 6 μg/ml of PHAwere included as controls. Co-cultures were incubated for 3 days andPBMC-proliferation was estimated using MTT reagent using the followingformula: % PBMC proliferation=[(OD570 of HFD/PBMC/PHA−OD570 ofHFD)/(OD570 of PBMC/PHA)]×10. As shown in FIG. 22, the HFD-mG1-GFP clone“mG1-R1” suppressed PBMC proliferation greater than controls and otherclones, indicating that exogenous HLA-G expression can provideimmunosuppression and/or reduced immunogenicity for differentiatedcells, such as fibroblasts.

NK-92 cytotoxicity Assays. The assays were performed substantially asdescribed previously. Briefly, 2.5×10³ target cells (i.e., HFD-m1-GFPcells or HFD-G0-GFP cells) were incubated with NK-92 cells at 3:1, 10:1,30:1 E:T ratio in CTL media for 7 hr. K562-WT cells were included aspositive control for NK-92 cytotoxicity. Cytotoxicity was determinedwith a CytoTox96 cytotoxicity assay kit The percentage of specific lysiswas determined as follows: % specific lysis=[(experimental LDHrelease−effector spontaneous release−target spontaneous release)/(targetmaximum release−target spontaneous release)]×100. As shown in Table 4below, the HFD-mG1-GFP clones “mG1-R1” and “mG1-#1” suppressed NK-92cytotoxicity greater than controls and other clones, indicating thatexogenous HLA-G expression can provide immunosuppression and/ordecreased immunogenicity for differentiated cells, such as fibroblasts.

TABLE 4 Cytotoxicity of NK-92 Against HFD-G0 and HFD-G1 Target CellsTarget cell % cytotoxicity (2.5 × 10³) 10:1 30:1 60:1 (E:T) HFD-G0-2 0 01.5 HFD-mG1-#1 0 1 4 HFD-mG1#3 0 0 0 HFD-mG1-R1 0 1 4 HFD-mG1-R2 2 1 9K562-WT X 36 44

1. A method of producing a genetically modified mammalian cell that hasreduced immunogenicity and/or improved immunosuppression as compared toa mammalian cell without said genetic modification, the methodcomprising: genetically modifying a mammalian cell with an exogenousnucleic acid comprising: a. a nucleic acid sequence encoding a Humanleukocyte antigen-G (HLA-G) protein comprising a full length amino acidsequence of SEQ ID NO:2, and operably linked to an Elongation Factor-1alpha (EF-1α) promoter comprising a sequence of SEQ ID NO:6; and b. a 3′untranslated region (UTR) comprising a full length nucleotide sequenceof SEQ ID NO:3; wherein the reduced immunogenicity and/or improvedimmunosuppression of the genetically modified cell is determined byeither: (1) a reduction of natural killer cell, NK-92 cytotoxicity ofthe genetically modified cell as compared to the mammalian cell withoutsaid genetic modification, (2) a reduction of in vitro peripheral bloodmononuclear cell proliferation of the genetically modified cell ascompared to the mammalian cell without said genetic modification, or (3)an increase in the size and weight of tumor formation by the geneticallymodified cell as compared to the mammalian cell without said geneticmodification in humanized NOD scid gamma, NSG mice.
 2. The method ofclaim 1, wherein the genetically modified mammalian cell expresses HLA-Gprotein for at least 7 weeks.
 3. The method of claim 1, wherein thegenetically modified mammalian cell expresses HLA-G protein for at least20 weeks.
 4. The method of claim 1, wherein the genetically modifiedmammalian cell expresses HLA-G protein for at least 50 weeks.
 5. Themethod of claim 1, wherein the genetically modified mammalian cellexpresses HLA-G protein on the cell surface of the genetically modifiedmammalian cell.
 6. The method of claim 1, wherein the geneticallymodified mammalian cell is a human cell.
 7. The method of claim 1,wherein the genetically modified cell is selected from the groupconsisting of a stem cell, a progenitor cell, a cell obtained by invitro differentiation of the stem cell or the progenitor cell, a fullydifferentiated cell, an epidermal progenitor cell, a pancreaticprogenitor cell, a hematopoietic stem cell, a keratinocyte, afibroblast, a hepatocyte, a mesenchymal stem cell, a cardiomyocyte, aneural stem cell, a neuron, and an astrocyte.
 8. The method of claim 1,wherein the genetically modified cell is selected from the groupconsisting of a human embryonic stem cell, a human mesenchymal stemcell, a human embryonic epidermal progenitor cell, and a human dermalfibroblast.
 9. The method of claim 1, wherein the exogenous nucleic acidis an expression vector.
 10. The method of claim 9, wherein theexpression vector is a transposon vector.
 11. The method of claim 9,wherein the expression vector further comprises a nucleic acid sequenceencoding a reporter protein.
 12. A method of producing an artificialtissue, a skin-graft, a skin-repair, or a skin-regeneration compositioncomprising the genetically modified mammalian cell produced by themethod of claim
 1. 13. The method of claim 1, wherein geneticallymodifying a mammalian cell with an exogenous nucleic acid comprisestransiently transfecting the mammalian cell with the exogenous nucleicacid.
 14. The method of claim 1, wherein genetically modifying amammalian cell with an exogenous nucleic acid comprises stablytransfecting the mammalian cell with the exogenous nucleic acid.
 15. Themethod of claim 1, wherein the genetically modified mammalian cells areselected using antibiotic resistance or flow cytometry.